Compositions and methods of use for mgd-csf in disease treatment

ABSTRACT

Disclosed is a newly identified secreted molecule, identified herein as “monocyte, granulocyte, and dendritic cell colony stimulating factor” (MGD-CSF), the polypeptide sequence, and polynucleotides encoding the polypeptide sequence. Also provided is a procedure for producing the polypeptide by recombinant techniques employing, for example, vectors and host cells. Additionally, procedures are described to modify the disclosed novel molecules of the invention to prepare fusion molecules. Also disclosed are methods for using the polypeptides and active fragments thereof for treatment of a variety of diseases, including, for example, cancer, autoimmune and inflammatory diseases, infectious diseases, and recurrent pregnancy loss.

PRIORITY CLAIM

This application claims priority to provisional applications 60/590,565,filed Jul. 22, 2004; 60/647,604, filed Jan. 27, 2005; 60/664,932, filedMar. 24, 2005; and a provisional application entitled “Novel MGD-CSFPolypeptides, Polynucleotides, and Methods of Use Thereof,” filed Jul.14, 2005. This application also relates to applications PCT/US03/34811,filed Oct. 31, 2003; PCT/US04/11270, filed Apr. 30, 2004; 60/590,565filed Jul. 22, 2004; 60/642,604, filed Jan. 11, 2005; 60/647,013, filedJan. 27, 2005; and 60/654,229, filed Feb. 18, 2005. These applicationsare all incorporated by reference in their entireties.

TECHNICAL FIELD

The present invention relates to a novel secreted molecule identifiedherein as “monocyte, granulocyte, and dendritic cell colony stimulatingfactor” (MGD-CSF). It relates to the polypeptide and polynucleotidesequences of MGD-CSF, fusion molecules containing MGD-CSF, vectors, hostcells, compositions, and kits comprising MGD-CSF, and methods of usingMGD-CSF and related molecules to diagnose, prevent, determine theprognosis for, and treat diseases, including immune-related diseases,infectious diseases, and cancer. MGD-CSF is a splice variant ofMCG34647, a gene with a previously unknown function.

BACKGROUND ART

Cells of the innate immune system, such as monocytes, macrophages,natural killer (NK) cells, and polymorphonuclear neutrophils (PMN), arethe first-line defenders against cancer and infectious disease by natureof their phagocytic, cytolytic, and antimicrobial properties. Monocytesand macrophages are believed to play an important role in inflammatorydiseases through their activation and secretion of inflammatorymediators. For example, granulocyte macrophage-colony stimulating factor(GM-CSF) is known to promote proliferation and differentiation ofgranulocytes, monocytes, and macrophages. Granulocyte-colony stimulatingfactor (G-CSF) is known to promote the differentiation and growth ofgranulocytes and neutrophils (Ogawa, Blood 81:3844-2853 (1993)). Atpresent, both G-CSF and GM-CSF are being used as protein therapeutics topromote the recovery of blood cells after chemotherapy, radiation, andbone marrow transplants.

NK cells are known to play a role in host responses to cancer. In bothsyngeneic and xenogenic transplant models, tumor Cells grow moreefficiently in NK−/− mice, and survival rates for the mice in thesemodels are significantly less than those for mice possessing NK cells.In addition, potentiating an NK response with soluble protein factors,such as IL-2 or IL-15, has been shown to increase the efficiency bywhich NK cells kill tumor cells in the presence of anti-tumor antibodiesboth in vitro and in vivo (Carson et al., J. Exp. Med. 180:1395-1403(1994)).

Additionally, NK cells are also known to play a role in host response toinfectious disease. For example, mice lacking NK cells are known to haveincreased susceptibility to viruses and intracellular pathogens.Similarly, humans with naturally occurring NK cell deficiencies are alsoknown to be highly susceptible to infections. In vitro, NK mediatedkilling of cells infected with virus or other intracellular pathogens isknown to be potentiated by cytokines such as interferon-α, interferon-β,interleukin-12, and interleukin-18 (Wu et al., Adv. Cancer Res. 90:127(2003)); Biron et al., Rev. Immunol. 17:189 (1999); Naume et al., Scand.J. Immunol. 40:128 (1994)).

It is also known that activated NK cells can be correlated with failurerates for women undergoing in vitro fertilization (IVF) procedures, andmay be further linked to spontaneous pregnancy loss (Dosiou et al.,Endocr. Rev. 26:44 (2005)). Additionally, elevated levels of activatedNK cells may be found in a number of patients with immune endometriosis,one known underlying cause of infertility in women (Dosiou et al.,Endocr. Rev. 26:44 (2005)).

Antigen-processing dendritic cells are capable of sensitizing T cells toboth new and recall antigens. Dendritic cells express high levels ofmajor histocompatibility complex class I and II antigens, which play arole in cancer immunotherapy, along with other immunomodulatoryproteins, adhesins, and cytokines. Dendritic cell cancer vaccines havebeen reported to be produced by extracting a patient's dendritic cellsand using immune cell stimulants to reproduce large amounts of dendriticcells in vitro or ex vivo. The dendritic cells can then be exposed toantigens from the patient's cancer cells. The combination of dendriticcells and antigens is injected into the patient, and the dendritic cellsprogram the patient's T cells. Dendritic cells break down the antigenson the cancer cell surfaces, then display them to killer T cells. (Songet al., Yonsei Med. J. 45 Suppl.:48-52 (2004)).

Cancer patients recovering from autologous hematopoietic celltransplantation exhibit decreased levels of circulating dendritic cells.Dendritic cells develop from hematopoietic progenitors and promotingtheir development may help regain normal dendritic cell levels. Theability to generate dendritic cells by inducing proliferation ofisolated human dendritic cells and inducing proliferation anddifferentiation of hematopoietic stem cells facilitates efficacy testsof dendritic cell vaccination and facilitates effective vaccinationpractice. There is a need in the art for factors that stimulatedendritic cell proliferation and/or hematopoietic stem cellproliferation and/or differentiation to dendritic cells. There is also aneed in the art for factors that promote the generation of dendriticcells from hematopoietic cells to increase circulating dendritic cellsin preparation for hematopoietic cell transplantation.

Osteoclasts share a common progenitor with dendritic cells, macrophages,and microglia (Servet-Delprat et al., BMC Immunol. 3:15 (2002)). Thesemultinucleated, adherent, bone-resorbing cells differentiate in the bonemarrow and function in the vicinity of the bone to regulate boneremodeling and calcium homeostasis. Osteoclast differentiation andfunction has been reported to be regulated by secreted factors,including M-CSF and osteoprotegerin (RANK ligand) (Miyamoto et al.,Keio. J. Med. 52:1-7 (2003)). Factors which play a role in theregulation of osteoclast differentiation and function may be therapeuticin treating osteoporosis and other bone diseases.

Microglial cells function as immune effectors of the central nervoussystem, where they also produce neurotrophic factors and regulateglutamate uptake. These mononuclear phagocytes are distributedthroughout the central nervous system parenchyma in both the white andgrey matter. Microglia have been reported to be present in increasednumbers in patients with Alzheimer's disease, wherein they displaymarked increases in nitric oxide production and inflammatory cytokines,including IL-1 and MIP1 alpha (Vincent et al. Neurobiol. Aging23:349-362 (2002)). Factors that regulate microglial differentiation andfunction may be therapeutic in treating Alzheimer's disease and otherneural diseases, including demyelinating diseases such as multiplesclerosis, acute disseminated encephalomyelopathy, progressivemultifocal leukoencephalopathy, stroke, and Parkinson's disease.

Gene MGC34647 encodes the hypothetical protein NP_(—)689669 (Strausberget al., Proc. Natl. Acad. Sci. 99:16,899 (2002)). The functions of thisgene and its encoded polypeptides are previously unknown. The sequencesof MGC34647 and NP_(—)689669 correspond to SEQ ID. NOS.: 49 and 103,respectively, of WO 2002/048337. They correspond to the amino acidsequence of a secreted protein of unknown function and its codingsequence, respectively.

INDUSTRIAL APPLICABILITY

Current therapies for immune diseases, cancer, infectious diseases, andimmune-mediated recurrent pregnancy loss are inadequate, insufficient,and often toxic. Novel therapeutic compounds and therapies that increasethe efficacy of the innate immune response to these conditions canprovide more efficient therapy and may have a better therapeutic indexthan current therapies.

BRIEF DESCRIPTION OF THE FIGURES AND TABLES Brief Description of theFigures

FIG. 1 shows the amino acid sequence alignment of exon 4 of MGD-CSF(MGD-CSF_exon4); SEQ ID NO:8), MGC34647 (NP_(—)689669); SEQ ID NO:10)and MGD-CSF SEQ ID NO:7), as further described in Example 1. Amino acididentity is designated by (*).

FIG. 2 shows a diagram of the pTT5 backbone vector used to generatepTT5-Gateway, which was used to transiently transfect mammalian cells,as further described in Example 2. FIG. 2 also shows the nucleic acidsequences of the multiple cloning site flanking sequences for the pTT5-G(Residues 1182-1265 and 2984-3127 of SEQ ID NO:272), pTT5-H, (Residues1182-1391 and 1597-1602 of SEQ ID NO:273, and pTT5-I (SEQ ID NO:284 andresidues 2905-3048 of SEQ ID NO:274 vectors.

FIG. 3 shows a diagram of the pTT2 backbone vector used to stablytransfect mammalian cells, as further described in Example 2.

FIG. 4 shows the expression of MGD-CSF with plasmid vectors on days 3through 6 post-transfection, as further described in Example 3 andTable 1. FIG. 4A shows the expression of intracellular (cells) andsecreted (supernatant) CLN00732663, a MGD-CSF vector with a C-terminalHis tag, in 293-6E cells. FIG. 4B shows the expression of intracellular(cells) and secreted (supernatant) CLN00816424, a MGD-CSF vector with aC-terminal His tag and a collagen leader sequence, in 293-6E cells. BothFIGS. 4A and 4B show Positope™ (Invitrogen, Carlsbad, Calif.) (rightpanels) as a positive control for expression. Molecular weights areindicated on the left panels.

FIG. 5 shows the degree of proliferation and the viability of cellstransfected with constructs described in Example 2 and Table 1 from days3 through 6 post-transfection, as further described in Example 4 andTable 1. FIG. 5A shows the degree of cell proliferation of cellstransfected with CLN00542945 (black bars), CLN00732663 (light greybars), CLN00821867 (diagonal stripe bars), and CLN00816424(cross-hatched bars), and compared to a control gene encoding secretedalkaline phosphatase (SEAP) (dark grey bars). Both the cells transfectedwith CLN00816424 and the control SEAP cells increased in number from 3through 6 days post-transfection, as further described in Example 4.FIG. 5B shows the percentage of viable cells transfected withCLN00542945 (black bars), CLN00732663 (light grey bars), CLN00821867(diagonal stripe bars), and CLN00816424 (cross-hatched bars), andcompared to a control gene encoding secreted alkaline phosphatase (SEAP)(dark grey bars). The cells transfected with CLN00816424 and the controlSEAP remained viable but the cells transfected with MGD-CSF,CLN00732663, and CLN00821867 demonstrated increased toxicity, which wasdependent on culture conditions, but was not gene-specific, and wasevidenced by their decreased viability over time in culture.

FIG. 6 shows that adherent 293-T cells expressing MGD-CSF can be adaptedto culture conditions with low serum concentrations, as furtherdescribed in Example 5. FIG. 6A shows the expression of MGD-CSF incultures of suspension 293-T cells grown in FreeStyle medium with 3% FBSand in HyQ-CHO medium with 1% FBS, as further described in Example 5.FIG. 6B shows the expression of MGD-CSF in suspension cultures with lowserum (panel 1), in the absence of serum (panel 2), and in adherentculture (panel 3), as compared to purified MGD-CSF standard producedfrom a bacterial host (panel 4).

FIG. 7 shows the expression of MGD-CSF in a bioreactor, as furtherdescribed in Example 6. Fermentation was monitored for 6 days. Sampleswere examined by gel electrophoresis and stained with Coomassie Blue ondays 1-6 post-inoculation. Molecular weights are indicated on the leftpanels. The position of MGD-CSF is indicated by the arrow. Increasingamounts of bovine serum albumin (BSA) is shown to quantify the amount ofprotein on the gel.

FIG. 8A shows the isolation of MGD-CSF from 293-T cells, as furtherdescribed in Example 7. Cell culture supernatant (Supt) was fractionatedon an SP-Sepharose FF column (SP-Pool), a Heparin Sepharose HP column(Hep-Pool), and a Q-Sepharose column (Q-Pool). MGD-CSF is glycosylatedand has an apparent molecular weight of 39 kDa by SDS-polyacrylamide gelelectrophoresis. Molecular weight markers are shown in the left lane.FIG. 8B shows the electrophoretic migration pattern of muteins ofMGD-CSF under reducing and non-reducing SDS-PAGE conditions, asdescribed in greater detail in Example 8. The left panel shows anSDS-PAGE gel run under conditions that reduce disulfide bonds and themiddle panel shows an SDS-PAGE gel run under conditions that do notreduce disulfide bonds. Wild type MGD-CSF expressed with its naturalsignal peptide (wt natSP), wild type MGD-CSF expressed with the collagensignal peptide (wt colSP), and the cysteine to serine muteins C35S,C167S, C176S, C178S, C179S, C190S, and C198S mutants are shown in bothpanels. Purified MGD-CSF is shown as a standard for quantitation andcomparison in the right panel. C179S and C190S were expressed at loweryields than wild type MGD-CSF, and C35S was not expressed at adetectable level.

FIG. 9 shows the ability of MGD-CSF to induce NK cell proliferationand/or survival, as further described in Example 9A. NK cell number wasexpressed in relative luciferase units (RLU) per well following exposureto a negative buffer control (diamonds) or conditioned media from cellstransfected with MGD-CSF (squares). MGD-CSF stimulated mouse NK cellproliferation. The control buffer had no effect. The proliferativeactivity of MGD-CSF on NK cells was specific and dose-dependent.

FIG. 10 shows the results of a screening assay for agents that induce NKcell proliferation and/or survival, as further described in Example 9A.The top and bottom panels represent two identical experiments, performedindependently. The number of human NK cells is expressed in relativeluciferase units (flu), following exposure to a test agent. Conditionedmedia from cells transfected with MGD-CSF or with plasmid DNA from thecluster 190647, the source of MGD-CSF, stimulated human NK cell survivaland/or proliferation. The positive controls IFNγ, IL-1, and GM-CSF alsostimulated NK cell proliferation.

FIG. 11 shows the ability of MGD-CSF to induce hematopoietic stem cellproliferation, as further described in Example 9B. The number of stemcells was determined by counting the cells with a hemocytometer. MGD-CSFincreased proliferation in a dose dependent manner. MGD-CSF (500 ng/ml)induced stem cell proliferation to a greater extent than M-CSF and to asimilar extent as G-CSF and GM-CSF.

FIG. 12 shows the ability of MGD-CSF to induce myelocytic cellproliferation, as further described in Example 9C. FIG. 12A compares theability of conditioned media containing MGD-CSF to induce myelocyteproliferation with the negative controls of the empty vector (Vector)and the irrelevant compounds CLN3732, FPT026, IL-10, and unconditionedmedia; and with the positive control GM-CSF. Monocyte number wasexpressed in relative luciferase units (RLU) per well following exposureto the test agent. FIG. 12B shows that both purified GM-CSF (stars) andconditioned media from cells transfected with MGD-CSF (squares)stimulated human monocyte proliferation. The control vector (diamonds)had no effect. The proliferative activity of MGD-CSF on monocytes wasspecific and dose-dependent.

FIG. 13 shows the results of a fluorescent activated cell sorting (FACS)analysis of granulocyte differentiation, as measured by the presence ofthe differentiation antigens CD67 and CD24 and described in greaterdetail in Example 10A. The number of hematopoeitic stem cells induced todifferentiate into granulocytes in response to media conditioned withnegative control vector (Vector CM) and media conditioned with theMGD-CSF vector (MGD-CSF CM) is shown. CD67 FITC (x-axis) indicates thenumber of CD67 positive cells by the fluorescence intensity of theantibody specific for CD67. CD24PE (y-axis) indicates the number of CD24positive cells by the fluorescence intensity of the antibody specificfor CD24. The outlined triangular area (arrow) indicates the number ofcells positive for both of the cell surface differentiation antigensCD67 and CD24 in each of the four panels. MGD-CSF CM stimulatedgranulocyte differentiation both in the absence (no cytokine) and thepresence of G-CSF. This stimulation by MGD-CSF was synergistic with theeffects of G-CSF.

FIG. 14 shows the results of a FACS analysis of granulocytedifferentiation, as measured by the presence of the differentiationantigens CD15 and CD24 and described in greater detail in Example 10A.CD24PE (x-axis) indicates the number of CD24 positive cells by thefluorescence intensity of the antibody specific for CD24. CD15APC(y-axis) indicates the number of CD15 positive cells by the fluorescenceintensity of the antibody specific for CD15. The box in the upper rightof each graph indicates the percent of cells that have both CD15 andCD34 differentiation markers on their cell surface. MGD-CSF inducedgranulocyte differentiation in a concentration-dependent manner, with adose of 500 ng/ml resulting in the differentiation of 8% of the bonemarrow hematopoeitic stem cells into granulocytes.

FIG. 15 shows the results of a FACS analysis of monocytedifferentiation, as measured by the presence of the differentiationantigens CD14 and CD3 and described in greater detail in Example 10B.The number of hematopoeitic stem cells induced to differentiate intomonoocytes in response to media conditioned with negative control vector(Vector CM) and media conditioned with the MGD-CSF vector (MGD-CSF CM)is shown. CD14FITC (x-axis) indicates the number of CD14 positive cellsby the fluorescence intensity of the antibody specific for CD14. CD3APC(y-axis) indicates the number of CD3 positive cells by the fluorescenceintensity of the antibody specific for CD3. The outlined oval area(arrow) indicates the number of cells positive for both of the cellsurface differentiation antigens CD14 and CD3 in each of the fourpanels. MGD-CSF CM stimulated monocyte differentiation. MGD-CSF CMstimulated monocyte differentiation both in the absence (no cytokine)and the presence (G-CSF) of G-CSF. The ability of MGD-CSF to inducemonocyte differentiation was greater than that of GM-CSF. MGD-CSF actedsynergistically with GM-CSF.

FIG. 16 shows the results of a FACS analysis of monocytedifferentiation, as measured by the presence of the differentiationantigens CD14 and CD16 and described in greater detail in Example 10B.CD14APC (y-axis) indicates the number of CD14 positive cells by thefluorescence intensity of the antibody specific for CD14. CD16FITC(x-axis) indicates the number of CD16 positive cells by the fluorescenceintensity of the antibody specific for CD16. The box in the upper rightof each graph indicates the percent of cells that have both CD14 andCD16 differentiation markers on their cell surface. MGD-CSF inducedmonocyte differentiation in a concentration-dependent manner, with adose of 100 ng/ml resulting in the differentiation of 10% of the bonemarrow hematopoeitic stem cells into monocytes.

FIG. 17 shows the results of a FACS analysis of dendritic celldifferentiation, as measured by the presence of the differentiationantigens CD86 and CD1 and described in greater detail in Example 10C.The number of hematopoeitic stem cells induced to differentiate intodendritic cells in response to media conditioned with negative controlvector (Vector CM) and media conditioned with the MGD-CSF vector(MGD-CSF) is shown. CD1aFITC (x-axis) indicates the number of CD1positive cells by the fluorescence intensity of the antibody specificfor CD1. CD86PE (y-axis) indicates the number of CD86 positive cells bythe fluorescence intensity of the antibody specific for CD86. Theoutlined oval area indicates the number of cells positive for both ofthe cell surface differentiation antigens CD1 and CD86. MGD-CSF CMstimulated dendritic cell differentiation. 4% of the cells transfectedwith the negative control differentiated into dendritic cells, while 22%of the cells transfected with the MGD-CSF vector differentiated intodendritic cells.

FIG. 18 shows the effect of MGD-CSF on human bone marrow colonyformation, as described in greater detail in Example 11. FIG. 18A showsthe dose-dependent stimulatory effect of MGD-CSF on the formation ofCFU-G (left panel) and CFU-M (right panel) in comparison to the effectsof G-CSF and GM-CSF. FIG. 18B shows the dose-dependent stimulatoryeffect of MGD-CSF on the formation of CFU-GM (left panel) and the totalcolony forming capacity (Total CFC) (right panel) in comparison to theeffects of G-CSF and GM-CSF. FIG. 18C shows the dose-dependentstimulatory effect of MGD-CSF on the formation of CFU-G (top leftpanel), CFU-GM (top right panel), CFU-M (bottom left panel), and thetotal colony forming capacity (Total CFC) (bottom right panel) incomparison to the effects of G-CSF and GM-CSF in the presence of thecytokines IL-3 and stem cell factor (SCF).

FIG. 19 shows a profile of the effect of MGD-CSF in assays for variousbiological activities, as described in greater detail in Example 12.MGD-CSF stimulated the proliferation of activated monocytes (MonPro4)and the proliferation of peripheral NK cells (NKGlo).

FIG. 20 shows a profile of the effect of MGD-CSF in various assays ofcytokine secretion, as described in greater detail in Example 13.MGD-CSF stimulated the secretion of GM-CSF (Luminex2-GMCSF), IL-2(Luminex2-IL2), and IL-13 (Luminex2-IL13).

FIG. 21 shows the effect of MGD-CSF on CFU-M formation, as described ingreater detail in Example 14. Examples of CFU-M formation induced bybuffer, GM-CSF, and G-CSF are shown in the top three panels. Thedose-dependent effect of CFU-M formation induced by 20 ng/ml, 100 ng/ml,and 500 ng/ml, MGD-CSF is shown in the bottom three panels.

FIG. 22 shows the effect of MGD-CSF on dendritic cell formation, asdescribed in greater detail in Example 15. Examples of dendritic cellformation induced by medium, 20 ng/ml MGD-CSF, 100 ng/ml MGD-CSF, and500 ng/ml MGD-CSF are shown. MGD-CSF induced the formation of elongateddifferentiated dendritic cells from spherical undifferentiatedhematopoeitic stem cells.

BRIEF DESCRIPTION OF THE TABLES

Table 1 provides information regarding SEQ. ID. NOS.:1-271, which arelisted in the Sequence Listing. Column 1 shows an internal designationidentification number (FP ID). Column 2 shows the nucleotide sequenceidentification number for the open reading frame of the nucleic acidsequence (SEQ. ID. NO.:(N1)). Column 3 shows the amino acid sequenceidentification number for the polypeptide sequence (SEQ. ID. NO.:(P1)).Column 4 shows the nucleotide sequence identification number for theentire nucleic acid sequence, including coding and noncoding regions(SEQ. ID. NO.:(N0)). Column 5 shows the corresponding nomenclature orthe NCBI accession number (Source ID). Column 6 shows the type ofsequence, for example, the function or vector composition (Type).

Table 2 annotates MGD-CSF with respect to NP_(—)689669, the publiclydisclosed sequence with the greatest degree of similarity. Row 1 showsan internal designation identification number (FP ID). Row 2 shows theclone identification number (Clone ID). Row 3 shows the predicted lengthof the polypeptide in number of amino acid residues (Pred Prot Len). Row4 shows the public accession identification number of a top human hitfound in the NCBI public database (Top Human Hit Accession No). Row 5shows the annotation of the top human hit set forth in row 4 (Top HumanHit Annotation). Row 6 shows the length of the top human hit in numberof amino acid residues (Top Human Hit Len). Row 7 shows the length ofthe match in number of amino acid residues between the query sequencedesignated by the FP ID and the top human hit (Match Len). Row 8 showsthe percent identity between the FP ID and the top human hit over thelength of the FP ID amino acid sequence expressed as a percentage (TopHuman Hit % ID over Query Len). Row 9 shows the percent identity betweenthe FP ID and the top human hit over the length of the top human hit (%ID over Hum Hit Len).

Table 3 shows the protein coordinates of MGD-CSF and NCBI NP_(—)689669.Row 1 shows an internal designation ID number of the polypeptide (FPID). Row 2 shows the clone identification number or NCBI accessionnumber of the polypeptide (Clone ID). Row 3 shows an internal clusteridentification number of the polypeptide (Cluster). Row 4 shows thatNP-689669 is secreted (Classification). Row 5 shows the predictedprotein length in number of amino acid residues (Pred Prot Len). Row 6shows an internal parameter predicting the likelihood that the FP ID issecreted with “1” being a high likelihood the polypeptide is secretedand “0” being a low likelihood of secretion (Treevote). Row 7 shows thelocation of the signal peptide coordinates (Signal Peptide Coords). Row8 shows the protein coordinates of the mature polypeptide with the firstamino acid residue at the N-terminus of the full-length polypeptidebeing amino acid number 1 (Mature Protein Coords).

Table 4 provides annotation for the secretory leader sequences shown inTable 1. Column 1 shows an internal designation ID number of thepolypeptide (FP ID). Column 2 shows the reference identification number(Source ID). Column 3 shows the NCBI annotation of the sequences.

Table 5 provides annotation for the MGD-CSF constructs shown Table 1.Column 1 shows the clone identification number (Clone ID). Column 2shows the NCBI annotation (Annotation). Column 3 lists the vector(Vector Description). Column 4 lists the tag, if any (Tag).

Table 6 shows the effect of MGD-CSF constructs on myelocytic cellproliferation in vitro as further described in Example 9C. Column 1shows the clone identification number (Clone ID). Column 2 shows theamino acid sequence of the clone. Column 3 provides a semiquantitativedescription of the potency of the activity of each clone to stimulatemonocyte proliferation (Potency). Column 4 provides a semiquantitativedescription of the degree of expression of each construct (Expression).

Table 7 shows the effect of MGD-CSF constructs on myelocytic cellproliferation in vivo. Table 7A shows the results of injecting a groupof six mice, three with a control vector and three with human MGD-CSFconstructs on myelocytic cell proliferation. Table 7B shows the resultsof injecting a group of twelve mice, six with a control vector and sixwith mouse MGD-CSF. In both Tables 7A and 7B, column 1 lists theidentification number of the animal (Animal ID), column 2 describes thevector as a control or MGD-CSF (Description), and column 3 indicates thenumber of monocytes in the peripheral blood (Monocytes/ul).

Table 8 shows the expression of the MGD-CSF gene as determined byinterrogating a GeneLogic database using Affymetrix U133 chip probes.Column 1 lists diseases in which MGC34647 was overexpressed (Disease).Column 2 lists specific pathologies associated with the diseases ofcolumn 1 (Pathology). Column 3 lists the number of disease specimensthat tested positive for the presence of MGC34647 (MGC34647 Positive).Column 4 lists the number of specimens examined (Total Gene Logic).Column 5 lists the percent of specimens examined which were positive (%Total). Columns 6 and 7 indicate that three acute promyelocytic leukemiasamples (13% of the total examined) were derived from the bone marrow.

SUMMARY

MGD-CSF promotes the proliferation, survival, and/or differentiation ofmonocytes, granulocytes, dendritic cells, and NK cells. Therefore,MGD-CSF finds use as a protein therapeutic for treating cancers throughits ability to stimulate the proliferation and/or activation of immunecells such as NK cells, monocytes, macrophages, granulocytes, anddendritic cells to fight tumor cells. MGD-CSF may be used alone or incombination with therapeutic monoclonal antibodies (for example,Rittman) to treat cancer, since MGD-CSF may promote antibody dependentcell cytotoxicity mediated by NK cells, monocytes, or granulocytes.MGD-CSF can also be used as an antagonistic therapeutic protein toeffect hematopoietic regeneration adjunctive to chemotherapy and bonemarrow transplantation. It can further be used to expand the number ofdendritic cells in vivo or ex vivo. In addition, MGD-CSF may be usefulas an anti-infectious agent in the treatment of infectious diseases,such as those caused by bacteria or viruses (for example, hepatitis Cvirus (HCV) or human immunodeficiency virus (HIV)).

MGD-CSF promotes the proliferation and/or the differentiation of immunecells, and thus finds use in treating immune diseases. It may play arole in the pathogenesis and treatment of autoimmune diseases. MGD-CSFantagonists may be developed as therapeutics for treating immunediseases. Antagonists may include monoclonal antibodies against MGD-CSF;MGD-CSF receptor(s), including soluble receptors; non-functionalmutants; antisense DNA; and RNAi.

The invention provides an isolated nucleic acid molecule comprising afirst polynucleotide that comprises a first nucleotide sequence chosenfrom SEQ. ID. NOS.:1, 2, 3, and 5; a first polynucleotide encoding afirst polypeptide comprising a first amino acid sequence chosen fromSEQ. ID. NOS.:7, 8, 9, and 11; a polynucleotide comprising a nucleotidesequence that is complementary to the first nucleotide sequence; and abiologically active fragment of any of these. In an embodiment, thebiologically active polypeptide fragment comprises at least sixcontiguous amino acid residues chosen from SEQ. ID. NOS.: 7, 8, 9, and11, and wherein at least two of the contiguous six amino acid residuesare leucine and arginine residues at amino acid residue positions 80 and81, respectively. This isolated nucleic acid molecule may be chosen froma cDNA molecule, a genomic DNA molecule, a cRNA molecule, a siRNAmolecule, an RNAi molecule, an mRNA molecule, an antisense molecule, anda ribozyme. In an embodiment, this nucleic acid molecule furthercomprises its complement.

In an embodiment, the first nucleotide sequence is SEQ. ID. NO.:3. Thisembodiment may further comprise a second polynucleotide. This secondpolynucleotide may comprise a second nucleotide sequence encoding ahomologous or heterologous secretory leader. The secretory leader may bechosen from SEQ. ID. NOS.:14-211.

The invention also provides a nucleic acid molecule at least about 70%,at least about 80%, or at least about 90% identical to the isolatednucleic acid molecule described above. The invention provides anisolated nucleic acid molecule that specifically hybridizes understringent conditions to the sequence set forth in SEQ. ID. NOS.:1, 2, or3, or to the complement of the sequence set forth in SEQ. ID. NOS.:1, 2,3, and 5, wherein the nucleic acid molecule encodes a polypeptide thatcan stimulate the proliferation and differentiation of granulocytes,monocytes, and dendritic cells.

The invention further provides an isolated polypeptide comprising afirst amino acid sequence chosen from SEQ. D. NOS.:7, 8, 9, and 11; asequence encoded by SEQ. ID. NOS.:1, 2, 3, and 5; and a biologicallyactive fragment of any of these. This isolated polypeptide may bepresent in a cell culture, for example, a bacterial cell culture, amammalian cell culture, an insect cell culture, or a yeast cell culture;or in a cell culture medium. This isolated polypeptide may also bepresent in a plant or a non-human animal.

In an embodiment, the biologically active fragment comprises at leastsix contiguous amino acid residues chosen from SEQ. ID. NOS.:7, 8, 9,and 11, wherein at least two of the contiguous six amino acid residuesare leucine and arginine at amino acid residue 80 and 81 of SEQ. ID.NO.:5.

The invention yet further provides an isolated polypeptide at leastabout 70%, at least about 80%, or at least about 90% homologous to anisolated polypeptide comprising a first amino acid sequence chosen fromSEQ. ID. NOS.:7, 8, 9, and 11; a sequence encoded by SEQ. ID. NOS.:1, 2,3, and 5; and a biologically active fragment of any of these.

The invention provides an isolated polypeptide comprising a first aminoacid sequence chosen from SEQ. ID. NOS.:7, 8, 9, and 11; a sequenceencoded by SEQ. ID. NOS.:1, 2, 3, and 5; and a biologically activefragment of any of these, further comprising a second amino acidsequence, wherein the second amino acid sequence is a homologoussecretory leader or a heterologous secretory leader, and wherein thefirst and second amino acid sequences are operably linked. Theheterologous leader sequence may be chosen from SEQ. ID. NOS.:14-211.

The invention also provides a vector comprising the nucleic acidmolecule described above and a promoter that regulates its expression.This vector may be a viral or a plasmid vector. The promoter may benaturally contiguous to the nucleic acid molecule or may not benaturally contiguous to the nucleic acid molecule. It may be aninducible promoter, a conditionally-active promoter, a constitutivepromoter, and/or a tissue-specific promoter.

Additionally, the invention provides a recombinant host cell comprisinga cell and one or more isolated nucleic acid, polypeptide, or vectordescribed above. The host cell may be prokaryotic or eukaryotic, forexample, a human, non-human mammalian, insect, fish, plant, or fungalcell. In an embodiment, the mammalian cell is of the CHO cell line orthe 293 cell line, for example, a 293T cell or a 293E cell.

The invention further provides a non-human animal injected with anisolated nucleic acid or polypeptide of the invention. This animal may,for example, be a rodent, a non-human primate, a rabbit, a dog, or apig.

The invention yet further provides a nucleic acid composition comprisingan isolated nucleic acid molecule of the invention and a carrier. Theinvention provides a polypeptide composition comprising an isolatedpolypeptide of the invention and a carrier. The invention provides avector composition comprising a vector of the invention and a carrier.The invention provides a host cell composition comprising a host cell ofthe invention and a carrier. In an embodiment, the carrier is apharmaceutically acceptable carrier.

In another aspect, the invention provides a method of producing arecombinant host cell comprising providing a vector that comprises anisolated nucleic acid molecule of the invention and allowing a cell tocome into contact with the vector to form a recombinant host celltransfected with the nucleic acid molecule.

The invention provides a method of producing a polypeptide comprisingproviding an isolated nucleic acid molecule of the invention andexpressing the nucleic acid molecule in an expression system to producethe polypeptide. In an embodiment, the expression system is a cellularexpression system, for example, a prokaryotic or a eukaryotic expressionsystem. This expression system may comprise a host cell transfected withan isolated nucleic acid molecule of the invention, forming arecombinant host cell, and further comprising culturing the recombinanthost cell to produce the polypeptide. In an embodiment, the expressionsystem is a cell-free expression system chosen from a wheat germ lysate,a rabbit reticulocyte, a ribosomal display, and an E. coli lysate.

The invention also provides a polypeptide produced by such a method. Forexample, it provides a polypeptide produced by a eukaryotic expressionsystem, as described above, wherein the host cell is chosen from amammalian cell, an insect cell, a plant cell, a yeast cell, and abacterial cell.

In yet another aspect, the invention provides a diagnostic kitcomprising a composition that comprises an isolated nucleic acidmolecule of the invention, a reporter for detecting the nucleic acidmolecule or its complement, and a vehicle. It provides a diagnostic kitcomprising an antibody that specifically binds to an isolatedpolypeptide of the invention and a carrier. It also provides adiagnostic kit comprising an isolated polypeptide of the invention and acarrier.

In a further aspect, the invention provides a method of determining thepresence of an antibody specific to an isolated polypeptide of theinvention in a patient sample comprising providing a compositioncomprising an isolated polypeptide of the invention, allowing thepolypeptide to interact with the sample; and determining whetherinteraction has occurred between the polypeptide and antibody in thesample, if present.

In yet a further aspect, the invention provides an isolated antibodythat specifically binds to and/or interferes with the activity of anantigen that comprises at least six contiguous amino acid residueschosen from SEQ. ID. NOS.:7-12. For example, these contiguous amino acidresidues may comprise the consecutive amino acid residues leu-arg atamino acid positions 80 and 81 of SEQ. ID. NO.:7 or the consecutiveamino acid residues leu-gln-arg of SEQ. ID. NO.:12. This antibody may bechosen from a polyclonal antibody, a monoclonal antibody, a single chainantibody, and active fragments of any of these, for example, an antigenbinding fragment, an Fc fragment, a cdr fragment, a V_(H) fragment, aV_(C) fragment, and a framework fragment.

The invention also provides an isolated polypeptide as described above,further comprising at least one fusion partner. By way of example, thefusion partner may be chosen from a polymer, a polypeptide, a succinylgroup, fetuin A, fetuin B, a leucine zipper domain, a tetranectintrimerization domain, a mannose binding protein, and an Fc region. Thepolymer may be a polyethylene glycol moiety, which may, for example,attach through an amino group of an amino acid of the polypeptide. Thispolyethylene glycol moiety may be a branched or linear chain polymer.

The invention further provides a method of screening for an agent thatmodulates activity of an isolated polypeptide of the inventioncomprising providing a test system in which an isolated polypeptide ofthe invention affects biological activity; and screening multiple agentsfor an effect on the activity of an isolated polypeptide of theinvention on the test system. The modulator may, for example, be a smallmolecule drug. The modulator may also, for example, be an antibody.

The invention further provides a method of stimulating immune cellscomprising providing a composition comprising a substantially purepolypeptide chosen from any of SEQ. ID. NOS.:7-12, and active fragmentsthereof; and contacting one or more immune cells with the polypeptide.The polypeptide may be encoded by a nucleic acid molecule comprising anucleotide sequence chosen from SEQ. ID. NOS.:1-6. Suitable immune cellsinclude granulocytes; monocytes; lymphocytes, such as NK cells;macrophages; peripheral blood mononuclear cells; and dendritic cells.

The invention yet further provides a method of increasing a populationof immune cells comprising providing a composition comprising asubstantially pure polypeptide chosen from SEQ. ID. NOS.:7-12 and activefragments of any of these; and contacting one or more immune cells orimmune cell precursors with the polypeptide. The polypeptide may beencoded by a nucleic acid molecule comprising a nucleotide sequencechosen from SEQ. M. NOS.:1-6. Suitable immune cell populations includepopulations of monocytes; lymphocytes, for example, NK cells;macrophages; and peripheral blood mononuclear cells.

The invention additionally provides a method of stimulating an immuneresponse in a subject comprising providing a composition comprising asubstantially pure polynucleotide encoding a polypeptide chosen fromSEQ. ID. NOS.:7-12 and active fragments of any of these; andadministering the composition to the subject. The polypeptide may beencoded by a nucleic acid molecule comprising a nucleotide sequencechosen from SEQ. ID. NOS.:1-6. The polypeptide may be administeredlocally or systemically. It may be administered intravenously, by enema,intraperitoneally, subcutaneously, topically, or transdermally.

The invention provides a method of increasing immune cells in a subjectundergoing cancer therapy comprising providing a composition comprisinga substantially pure polypeptide chosen from any of SEQ. ID. NOS.:7-12and active fragments of any of these; and administering the compositionto the subject. The polypeptide may be encoded by a nucleic acidmolecule comprising a nucleotide sequence chosen from SEQ. ID. NOS.:1-6.Suitable immune cell populations include populations of monocytes;lymphocytes, for example, NK cells; macrophages; and peripheral bloodmononuclear cells. The cancer therapy is may comprise chemotherapyand/or radiation therapy. The polypeptide may be administered after abone marrow transplant.

The invention also provides a method of treating or preventing cancer ina subject comprising providing a composition comprising a substantiallypure polypeptide chosen from SEQ. ID. NOS.:7-12 and active fragments ofany of these; and administering the composition to the subject.

The invention further provides a method for inhibiting tumor growth in asubject comprising providing a composition comprising a substantiallypure polypeptide chosen from SEQ. ID. NOS.:7-12 and active fragments ofany of these; and administering the composition to the subject. Thetumor may comprise human tumor cells, for example, solid tumor cells orleukemic tumor cells.

The invention yet further provides a method of treating or preventing aninfection in a subject comprising providing a composition comprising asubstantially pure polypeptide chosen from SEQ. ID. NOS.:7-12 and activefragments of any of these; and administering the composition to thesubject. The polypeptide may be encoded by a nucleic acid moleculecomprising a nucleotide sequence chosen from SEQ. ID. NOS.:1-6. Thismethod can, for example, treat or prevent a bacterial infection, amycoplasma infection, a fungal infection, a viral infection, anintracellular pathogen, and/or an intracellular parasite. The method maybe practiced by administering the composition to the subject locally orsystemically.

Additionally, the invention provides a method for modulating an immuneresponse in a subject, comprising providing a modulator of a polypeptidechosen from SEQ. ID. NOS.:7-12 and active fragments of any of these; andadministering the modulator to the subject. The modulator may, forexample, be an antibody, a soluble receptor, and/or a polypeptide.Suitable antibody modulators include monoclonal antibodies, polyclonalantibodies, cdr fragments, V_(H) fragments, V_(C) fragments, frameworkfragments, single chain antibodies, and active fragments of an antibody.The modulator may also, for example, be an aptamer, an RNAi, anantisense molecule, and/or a ribozyme. The method can, for example,modulate the immune response by suppressing inflammation and/orautoimmune disease. The method can also, for example, modulate theimmune response by treating or preventing rheumatoid arthritis,osteoarthritis, psoriasis, inflammatory bowel disease, multiplesclerosis, myocardial infarction, stroke, and/or fulminant liverfailure.

The invention provides a method of modulating an immune response topregnancy comprising providing a modulator of a polypeptide chosen fromSEQ. ID. NOS.:7-12 and active fragments of any of these; andadministering the modulator to the subject. The method can, for example,by reduce recurrent pregnancy loss, modulating the immune response.

The invention provides a method of enhancing immune response to avaccine in a subject comprising providing a polypeptide compositioncomprising a substantially purified polypeptide chosen from SEQ. ID.NOS.:7-12 and active fragments of any of these; providing a vaccinecomposition; and administering the polypeptide composition and thevaccine composition to the subject. The polypeptide composition may beadministered to the subject prior to, substantially contemporaneouslywith, or after administering the vaccine composition.

The invention also provides a method of treating or preventing aninflammatory disease in a subject comprising providing a modulator of apolypeptide chosen from SEQ. ID. NOS.:7-12 and active fragments of anyof these; and administering the modulator to the subject. The modulatormay, for example, be an aptamer, an RNAi, an antisense molecule, and/ora ribozyme. The modulator may also, for example, be an antibody, asoluble receptor, and/or a polypeptide. Suitable antibody modulatorsinclude monoclonal antibodies, polyclonal antibodies, cdr fragments,V_(H) fragments, V_(C) fragments, framework fragments, single chainantibodies, and active fragments of an antibody.

The invention further provides a method of treating or preventing anautoimmune disease in a subject comprising providing a modulator of apolypeptide chosen from SEQ. ID. NOS.:7-12 and active fragments of anyof these; and administering the modulator to the subject. The modulatormay, for example, be an aptamer, an RNAi, an antisense molecule, and/ora ribozyme. The modulator may also, for example, be an antibody, asoluble receptor, and/or a polypeptide. Suitable antibody modulatorsinclude monoclonal antibodies, polyclonal antibodies, cdr fragments,V_(H) fragments, V_(C) fragments, framework fragments, single chainantibodies, and active fragments of an antibody.

The invention yet further provides a method of increasing the number ofNK cells in a subject comprising providing a polypeptide chosen fromSEQ. ID. NOS.:7-12 and active fragments of any of these; andadministering the polypeptide to the subject.

The invention provides a method of modulating an NK cell population in asubject comprising providing a modulator of a polypeptide chosen fromSEQ. ID. NOS.:7-12 and active fragments of any of these; andadministering the modulator to the subject. The modulator may, forexample, be an aptamer, an RNAi, an antisense molecule, and/or aribozyme. The modulator may also, for example, be an antibody, a solublereceptor, and/or a polypeptide. Suitable antibody modulators includemonoclonal antibodies, polyclonal antibodies, cdr fragments, V_(H)fragments, V_(C) fragments, framework fragments, single chainantibodies, and active fragments of an antibody. In an embodiment, theNK cell population stimulates an immune response. In an embodiment, theNK cell population suppresses pregnancy loss. In an embodiment, the NKcell population stimulates an anti-cancer response.

The invention also provides a method of increasing a population ofhematopoietic stem cells comprising providing a composition comprising asubstantially pure polypeptide chosen from SEQ. ID. NOS.:7-12 and activefragments of any of these; and contacting the population ofhematopoietic stem cells with the polypeptide.

The invention further provides a method of providing cytoprotection to apopulation of cells comprising providing a composition comprising asubstantially pure polypeptide chosen from SEQ. ID. NOS.:7-12 and activefragments of any of these; and contacting the population of cells withthe polypeptide.

The methods for modulating or enhancing immune responses, treating orpreventing disease, increasing the number of NK cells, modulating an NKcell population, increasing a population of hematopoietic stem cells,and providing cytoprotection may be practiced, for example, byadministering the compositions described above locally or systemically.They may also be practiced by providing the polypeptide compositionsdescribed above, wherein the polypeptide is encoded by a nucleic acidmolecule comprising a nucleotide sequence chosen from SEQ. ID. NOS.:1-6and active fragments of any of these. They may further be practiced byproviding the polypeptide compositions described above, wherein thepolypeptide further comprises at least one fusion partner. By way ofexample, the fusion partner may be chosen from a polymer, a polypeptide,a succinyl group, fetuin A, fetuin B, a leucine zipper domain, atetranectin trimerization domain, a mannose binding protein, and an Fcregion. The polymer may be a polyethylene glycol moiety, which may, forexample, attach through an amino group of an amino acid of thepolypeptide. This polyethylene glycol moiety may be a branched or linearchain polymer.

DESCRIPTION OF EMBODIMENTS Definitions

The terms used herein have their ordinary meanings, as set forth below,and can be further understood in the context of the specification.

“Monocyte, granulocyte, and dendritic cell colony stimulating factor”(MGD-CSF) is a novel, isolated, secreted molecule having the nucleicacid and amino acid sequences shown as SEQ. ID. NOS.:1, and 7,respectively. Provisional applications 60/590,565 and 60/564,932referred to MGD-CSF as FPT025. The term “molecules of the invention” isused herein to include any of SEQ ID NOS.:1-13, any of SEQ ID NOS.:1-13with a secretory leader of any of SEQ. ID NOS.:14-211, and any of theconstructs of SEQ. ID NOS.:212-271.

The terms “nucleic acid molecule,” “nucleotide,” “polynucleotide,” and“nucleic acid” are used interchangeably herein to refer to polymericforms of nucleotides of any length. They can include both double- andsingle-stranded sequences and include, but are not limited to, cDNA fromviral, prokaryotic, and eukaryotic sources; mRNA; genomic DNA sequencesfrom viral (e.g. DNA viruses and retroviruses) or prokaryotic sources;RNAi; cRNA; antisense molecules; ribozymes; and synthetic DNA sequences.The term also captures sequences that include any of the known baseanalogs of DNA and RNA.

“Recombinant,” as used herein to describe a nucleic acid molecule, meansa polynucleotide of genomic, cDNA, viral, semisynthetic, and/orsynthetic origin which, by virtue of its origin or manipulation, is notassociated with all or a portion of the polynucleotide with which it isassociated in nature. The term “recombinant” as used with respect to aprotein or polypeptide, means a polypeptide produced by expression of arecombinant polynucleotide. The term “recombinant” as used with respectto a host cell means a host cell into which a recombinant polynucleotidehas been introduced.

A “complementary” nucleotide sequence acid molecule is a one that iscomprised of its base pair complements. Deoxyribonucleotides with thebase adenine are complementary to those with the base thymidine, anddeoxyribonucleotides with the base thymidine are complementary to thosewith the base adenine. Deoxyribonucleotides with the base cytosine arecomplementary to those with the base guanine, and deoxyribonucleotideswith the base guanine are complementary to those with the base cytosine.Ribonucleotides with the base adenine are complementary to those withthe base uracil, and deoxyribonucleotides with the base uracil arecomplementary to those with the base adenine. Ribonucleotides with thebase cytosine are complementary to those with the base guanine, anddeoxyribonucleotides with the base guanine are complementary to thosewith the base cytosine.

A “promoter,” as used herein, is a DNA regulatory region capable ofbinding RNA polymerase in a mammalian cell and initiating transcriptionof a downstream (3′ direction) coding sequence operably linked thereto.For purposes of the present invention, a promoter sequence includes theminimum number of bases or elements necessary to initiate transcriptionof a gene of interest at levels detectable above background. Within thepromoter sequence may be a transcription initiation site, as well asprotein binding domains (consensus sequences) responsible for thebinding of RNA polymerase. Eukaryotic promoters will often, but notalways, contain “TATA” boxes and “CAT” boxes. Promoters include thosethat are naturally contiguous to a nucleic acid molecule and those thatare not naturally contiguous to a nucleic acid molecule. Additionally,the term “promoter” includes inducible promoters, conditionally activepromoters such as a cre-lox promoter, constitutive promoters, and tissuespecific promoters.

“Operably linked” refers to an arrangement of elements wherein thecomponents so described are configured so as to perform their desiredfunction. Thus, a secretory leader sequence operably linked to apolypeptide sequence is capable of effecting the secretion of thepolypeptide from the cell.

“Transfected” means possessing introduced DNA or RNA, with or withoutthe use of any accompanying facilitating agents such as lipofectamine.Methods for transfection that are known in the art include calciumphosphate transfection, DEAE dextran transfection, protoplast fusion,electroporation, and lipofection.

“Expression of a nucleic acid molecule” refers to the conversion of theinformation contained in the nucleic acid molecule into a gene product.The gene product can be the direct transcriptional product of a gene(e.g., mRNA, tRNA, rRNA, antisense RNA, ribozyme, structural RNA, or anyother type of RNA) or a peptide or polypeptide produced by translationof an mRNA. Gene products also include RNAs which are modified byprocesses such as capping, polyadenylation, methylation, and editing;and proteins modified by, for example, methylation, acetylation,phosphorylation, ubiquitination, ADP-ribosylation, myristilation, andglycosylation.

A “vector” is an agent, typically a virus or a plasmid, which can beused to transfer genetic material to a cell or organism.

A “host cell” is an individual cell or a cell culture which can be orhas been a recipient of any recombinant vector(s) or isolatedpolynucleotide(s). Host cells include progeny of a single host cell, andthe progeny may not necessarily be completely identical (in morphologyor in total DNA complement) to the original parent cell due to natural,accidental, or deliberate mutation and/or change. A host cell includescells transfected or infected in vivo or in vitro with a recombinantvector or a polynucleotide of the invention. A host cell which comprisesa recombinant vector of the invention may be called a “recombinant hostcell.”

A “stem cell” is an undifferentiated pluripotent or multipotent cellwith the ability to self-renew, to remain undifferentiated, and tobecome differentiated. Stem cells can divide without limit, at least forthe lifetime of the animal in which they naturally reside. Stem cellsare not terminally differentiated, meaning they are not at the end of adifferentiation pathway. When a stem cell divides, each daughter cellcan either remain a stem cell or it can embark on a course that leads toterminal differentiation. A stem cell can be an embryonic stem cell, ajuvenile stem cell, or an adult stem cell. A “hematopoeitic stem cell”is involved in the process of hematopoeisis, which is the process offorming mature blood cells from precursor cells.

The terms “polypeptide” and “protein” are used interchangeably to referto a polymer of amino acid residues, and are not limited to a minimumlength. Thus, peptides, oligopeptides, dimers, multimers, and the like,are included within the definition. Both full-length proteins andfragments thereof are encompassed by the definition. The terms alsoinclude post-expression modifications of the polypeptide, for example,glycosylation, acetylation, phosphorylation, and the like. Furthermore,for purposes of the present invention, a “polypeptide” refers to aprotein which includes modifications, such as deletions, additions, andsubstitutions (generally conservative in nature), to the nativesequence, as long as the protein maintains the desired activity. Thesemodifications may be deliberate, as through site-directed mutagenesis,or may be accidental, such as through mutations of hosts which producethe proteins or errors due to PCR amplification.

A “leader sequence” comprises a sequence of amino acid residues,beginning at amino acid residue 1, which is located at the aminoterminus of the polypeptide, and extending to a cleavage site, which,upon proteolytic cleavage, results in formation of a mature protein.Leader sequences are generally hydrophobic and have some positivelycharged residues. Leader sequences can be natural or synthetic,heterologous, or homologous with the protein to which they are attached.A “secretory leader” is a leader sequence that directs a protein to besecreted from the cell.

A “fusion partner” is a polypeptide fused in-frame at the N-terminusand/or C-terminus of a therapeutic or prophylactic polypeptide, orinternally to a therapeutic or prophylactic polypeptide.

The term “receptor” refers to a polypeptide that binds to a specificligand. The ligand is usually an extracellular molecule which, uponbinding to the receptor, usually initiates a cellular response, such asinitiation of a signal transduction pathway. A “soluble receptor” is areceptor that lacks a membrane anchor domain, such as a transmembranedomain. A “soluble receptor” may include naturally occurring splicevariants of a wild-type transmembrane protein receptor in which thetransmembrane domain is spliced out. A “soluble receptor” may includethe extracellular domain or any fragment of the extracellular domain ofa transmembrane protein receptor. Soluble receptors can modulate atarget protein. They can, for example, compete with wild-type receptorsfor ligand binding and participate in ligand/receptor interactions, thusmodulating the activity of or the number of the receptors and/or thecellular activity downstream from the receptors. This modulation maytrigger intracellular responses, for example, signal transduction eventswhich activate cells, signal transduction events which inhibit cells, orevents that modulate cellular growth, proliferation, differentiation,and/or death, or induce the production of other factors that, in turn,mediate such activities.

An “isolated,” “purified,” “substantially isolated,” or “substantiallypure” molecule (such as a polypeptide or polynucleotide) is one that hasbeen manipulated to exist in a higher concentration than in nature. Forexample, a subject antibody is isolated, purified, substantiallyisolated, or substantially purified when at least 10%, or 20%, or 40%,or 50%, or 70%, or 90% of non-subject-antibody materials with which itis associated in nature have been removed. As used herein, an“isolated,” “purified,” “substantially isolated,” or “substantiallypurified” molecule includes recombinant molecules.

A “biologically active” entity, or an entity having “biologicalactivity,” is one having structural, regulatory, or biochemicalfunctions of a naturally occurring molecule or any function related toor associated with a metabolic or physiological process. Biologicallyactive polynucleotide fragments are those exhibiting activity similar,but not necessarily identical, to an activity of a polynucleotide of thepresent invention. The biological activity can include an improveddesired activity, or a decreased undesirable activity. For example, anentity demonstrates biological activity when it participates in amolecular interaction with another molecule, such as hybridization, whenit has therapeutic value in alleviating a disease condition, when it hasprophylactic value in inducing an immune response, when it hasdiagnostic and/or prognostic value in determining the presence of amolecule, such as a biologically active fragment of a polynucleotidethat can, for example, be detected as unique for the polynucleotidemolecule, or that can be used as a primer in a polymerase chainreaction. A biologically active polypeptide or fragment thereof includesone that can participate in a biological reaction, including, but notlimited to, one that can serve as an epitope or immunogen to stimulatean immune response, such as production of antibodies; or that canparticipate in modulating the immune response.

The terms “antibody” and “immunoglobulin” are used interchangeably torefer to a protein, for example, one generated by the immune system,synthetically, or recombinantly, that is capable of recognizing andbinding to a specific antigen. Antibodies are commonly known in the art.Antibodies may recognize polypeptide or polynucleotide antigens. Theterm includes active fragments, including for example, an antigenbinding fragment of an immunoglobulin, a variable and/or constant regionof a heavy chain, a variable and/or constant region of a light chain, acomplementarity determining region (cdr), and a framework region. Theterms include polyclonal and monoclonal antibody preparations, as wellas preparations including hybrid antibodies, altered antibodies,chimeric antibodies, hybrid antibody molecules, F(ab)₂ and F(ab)fragments; Fv molecules (for example, noncovalent heterodimers), dimericand trimeric antibody fragment constructs; minibodies, humanizedantibody molecules, and any functional fragments obtained from suchmolecules, wherein such fragments retain specific binding.

A “vaccine” is a preparation that produces or artificially increasesimmunity to a particular disease. It may, for example, be comprised ofkilled microorganisms, living attenuated organisms, or living virulentorganisms that is administered to produce or artificially increaseimmunity to a particular disease. It includes a preparation containingweakened or dead microbes of the kind that cause a particular disease,administered to stimulate the immune system to produce antibodiesagainst that disease. The term includes nucleic acid and polypeptidevaccines.

The term “binds specifically,” in the context of antibody binding,refers to high avidity and/or high affinity binding of an antibody to aspecific epitope. Hence, an antibody that binds specifically to oneepitope (a “first epitope”) and not to another (a “second epitope”) is a“specific antibody.” An antibody specific to a first epitope may crossreact with and bind to a second epitope if the two epitopes sharehomology or other similarity. The term “binds specifically,” in thecontext of a polynucleotide, refers to hybridization under stringentconditions. Conditions that increase stringency of both DNA/DNA andDNA/RNA hybridization reactions are widely known and published in theart (Curr. Prot. Molec. Biol., John Wiley & Sons (2001)).

The terms “subject,” “individual,” “host,” and “patient” are usedinterchangeably herein to refer to a living animal, including a humanand a non-human animal. The subject may, for example, be an organismpossessing immune cells capable of responding to antigenic stimulation,and stimulatory and inhibitory signal transduction through cell surfacereceptor binding. The subject may be a mammal, such as a human ornon-human mammal, for example, dogs, cats, pigs, cows, sheep, goats,horses, rats, and mice. The term “subject” does not preclude individualsthat are entirely normal with respect to a disease, or normal in allrespects.

A “patient sample” is any biological specimen derived from a patient.The term includes, but is not limited to, biological fluids such asblood, serum, plasma, urine, cerebrospinal fluid, tears, saliva, lymph,dialysis fluid, lavage fluid, semen, and other liquid samples, as wellas cell and tissues of biological origin. The term also includes cellsor cells derived therefrom and the progeny thereof, including cells inculture, cell supernatants, and cell lysates. It further includes organor tissue culture-derived fluids, tissue biopsy samples, tumor biopsysamples, stool samples, and fluids extracted from physiological tissues,as well as cells dissociated from solid tissues, tissue sections, andcell lysates. This definition encompasses samples that have beenmanipulated in any way after their procurement, such as by treatmentwith reagents, solubilization, or enrichment for certain components,such as polynucleotides or polypeptides. Also included in the term arederivatives and fractions of patient samples. A patient sample may beused in a diagnostic, prognostic, or other monitoring assay.

A “disease” is a pathological condition, for example, one that can beidentified by symptoms or other identifying factors as diverging from ahealthy or a normal state. The term “disease” includes disorders,syndromes, conditions, and injuries. Diseases include, but are notlimited to, proliferative, inflammatory, immune, metabolic, infectious,and ischemic diseases.

The term “modulate” refers to the production, either directly orindirectly, of an increase or a decrease, a stimulation, inhibition,interference, or blockage in a measured activity when compared to asuitable control. A “modulator” of a polypeptide or polynucleotide or an“agent” are terms used interchangeably herein to refer to a substancethat affects, for example, increases, decreases, stimulates, inhibits,interferes with, or blocks a measured activity of the polypeptide orpolynucleotide, when compared to a suitable control.

“Preventing,” as used herein, includes providing prophylaxis withrespect to the occurrence or recurrence of a disease in a subject thatmay be predisposed to the disease but has not yet been diagnosed withthe disease. Treatment and prophylaxis can be administered to anorganism, including a human, or to a cell in vivo, in vitro, or ex vivo,and the cell subsequently administered the subject.

“Treatment,” as used herein, covers any administration or application ofremedies for disease in a mammal, including a human, and includesinhibiting the disease. It includes arresting disease development andrelieving the disease, such as by causing regression or restoring orrepairing a lost, missing, or defective function, or stimulating aninefficient process.

A “carrier” refers to a solid, semisolid or liquid filler, diluent,encapsulating material, formulation auxiliary, or excipient of anyconventional type. A “pharmaceutically acceptable carrier” refers to anon-toxic “carrier.” A pharmaceutically acceptable carrier is non-toxicto recipients at the dosages and concentrations employed and iscompatible with other ingredients of the formulation. Pharmaceuticallyacceptable carriers can be, for example, vehicles, adjuvants, ordiluents.

MGD-CSF and Related Nucleic Acids and Polypeptides

The invention provides a novel isolated secreted molecule, identifiedherein as “monocyte, granulocyte, and dendritic cell colony stimulatingfactor” (MGD-CSF). The invention provides methods of using MGD-CSF, aswell as related factors, which include variants and mutants of MGD-CSF.MGD-CSF is related to NP_(—)689669, Molecular Genomics Clone MGC34647,and Incyte SEQ. ID. NOS.:232, 255 and 257 (WO 2002/048337), as furtherdescribed below.

MGD-CSF is 241 amino acids in length and comprises a signal peptide orsecretory leader sequence. MGD-CSF is a subclone derived from the motherclone CLN00506579, in clone family CLN00212388. MGD-CSF belongs to FivePrime's cluster 190647. This cluster of secreted proteins includes allexpressed sequences representing a single gene. Its status as a secretedmolecule is confirmed by its Treevote of 0.92. The Treevote is theresult of an algorithm constructed on the basis of a number of physicaland chemical attributes that predicts whether a predicted amino acidsequence is secreted; a Treevote greater than 0.50 is indicative of asecreted molecule.

As shown in FIG. 1, MGD-CSF is related to a hypothetical proteinpredicted to be encoded by the mRNA sequence of NP_(—)689669, asdesignated by the National Center for Biotechnology Information (NCBI)(Strausberg et al., Proc. Natl. Acad. Sci. 99:16,899 (2002)). Thishypothetical human protein is predicted to comprise 242 amino acids. Thecoding sequence for NP 689669 has been described by the NationalInstitutes of Health's Mammalian Gene Collection (MGC) as MCG34647. Thenucleic acid sequences of MGC34647 correspond to SEQ ID. NOS.:49 and103, respectively, as designated in WO 2002/048337, wherein SEQ. ID.NO.:49 was described as secreted protein of unknown function, encoded bySEQ. ID. NO.:103. The functions of MGC34647 and NP 689669 wereheretofore undisclosed. MGD-CSF is a novel splice variant of MGC34647.The junction between exon 3 and exon 4 is differentially spliced suchthat amino acid L80 is followed by R81.

Gene MGC34647 is predicted to encode a protein with an open readingframe of 242 amino acids, with a nucleic acid coding sequence 729nucleotides in length. The proprotein is predicted to weigh 27,479daltons and have an isoelectric point of 7.72. Following cleavage of asignal peptide, which comprises amino acids 1-20, the mature protein ispredicted to weigh 25,229 daltons and have an isoelectric point of 6.74.This mature protein is predicted to be 222 amino acids long, encoded bya nucleic acid molecule of 669 nucleotides. MGC34647 has six exons. Itmaps to the genome on chromosome 16q22.1 from the start position of70456649 to the stop position of 70470765.

MGC34647 expression has been observed in spleen, parotid gland, jointmeniscus, bile duct, seminal vesicle, medulla oblongata, pituitarygland, salivary gland, and the Sequence Listing, as further describedbelow. Based on these localizations, MGC34647 is predicted to haveseveral specific therapeutic uses. It may be the target of anantagonistic antibody for autoimmune diseases, for example, multiplesclerosis, rheumatoid arthritis, and systemic lupus erythematosus (SLE).It may, for example, be used as a protein therapeutic agonist forhematopoietic cell regeneration during chemotherapy and bone marrowtransplant, as an antagonistic protein therapeutic to enhancecell-mediated immunity for treating infectious diseases, or as a proteintherapeutic antagonist for cytoprotection.

Nucleic Acids

The present invention provides nucleic acid molecules comprising apolynucleotide sequence corresponding to the novel MGD-CSF sequences asset forth in the Tables and Sequence Listing, for example, SEQ. ID.NOS.:1, 2, 3, and 5. The invention provides uses for these nucleic acidmolecules, and for related nucleic acid molecules, such as those shownin SEQ. ID. NOS.:4 and 13. These uses are described herein.

The invention provides a DNA molecule that contains a promoter of aliver-expressed gene operably linked to a gene encoding MGD-CSF orNP_(—)688669, and that can be expressed in vivo to produce a proteinthat is functionally active. DNA molecules as described have a varietyof uses, for example as tools in basic research to study the in vivofunction of an artificially introduced MGD-CSF or NP_(—)688669, theinteraction of more than one artificially introduced MGD-CSF orNP_(—)688669, the in vivo dynamics of artificially introduced MGD-CSF orNP_(—)688669 fusion proteins, or to identify the in vivo targets of anartificially introduced MGD-CSF or NP_(—)688669 protein, and astherapeutic treatments, as further described below.

Non-limiting embodiments of nucleic acid molecules include genes or genefragments, exons, introns, mRNA, tRNA, rRNA, siRNA, ribozymes, antisensecDNA, recombinant polynucleotides, branched polynucleotides, plasmids,vectors, isolated DNA of any sequence, isolated RNA of any sequence,nucleic acid probes, and primers. Nucleic acid molecules include splicevariants of an mRNA. Nucleic acids can be naturally occurring, forexample DNA or RNA, or can be synthetic analogs, as known in the art.Such analogs demonstrate stability under assay conditions, thus they aresuitable as probes. A nucleic acid molecule can also comprise modifiednucleic acid molecules, such as methylated nucleic acid molecules andnucleic acid molecule analogs. Analogs of purines and pyrimidines areknown in the art.

Nucleic acids of the invention are useful as hybridization probes fordifferential identification of the tissue(s) or cell type(s) present ina biological sample. Fragments of the full length MGD-CSF variant may beused as hybridization probes for cDNA libraries to isolate the fulllength gene and to isolate other genes which have a high sequencesimilarity or a similar biological activity. Probes of this type canhave at least 30 bases and may comprise, for example, 50 or more bases.The probe may also be used in a screening procedure to identify a cDNAclone corresponding to a full length transcript and a genomic clone orclones that contain complete MGD-CSF genes, including regulatory andpromoter regions, exons, and introns. An example of such a screen wouldinclude isolating the coding regions of MGD-CSF genes by using a knownnucleic acid sequence to synthesize an oligonucleotide probe. Labeledoligonucleotides having a sequence complementary to a gene of thepresent invention can be used to screen a human cDNA, a genomic DNA, ora mRNA library to identify complementary library components.

The present invention further relates to polynucleotides which hybridizeto the hereinabove-described sequences if there is at least 91%, atleast 92%, or at least 95% identity between the sequences. The presentinvention relates to polynucleotides which hybridize under stringentconditions to the hereinabove-described polynucleotides. Stringentconditions generally include condition under which hybridization willoccur only if there is at least 95%, or at least 97% identity betweenthe sequences. For example, overnight incubation at 42° C. in a solutioncontaining 50% formamide, 5×SSC (150 mM NaCl, 15 mM trisodium citrate),50 mM sodium phosphate (pH 7.6), 5×Denhardt's solution, 10% dextransulfate, and 20 μg/ml denatured, sheared salmon sperm DNA, followed bywashing the filters in 0.1×SSC at about 65° C., constitute stringentconditions.

The polynucleotides which hybridize to the polynucleotides shown in theTables and Sequence Listing can encode polypeptides which retainsubstantially the same biological function or activity as the maturepolypeptide. Alternatively, a polynucleotide may have at least 20 bases,at least 30 bases, or at least 50 bases which hybridize to apolynucleotide of the present invention and which has an identitythereto, and which may or may not retain the same biological function oractivity as the mature polypeptide. Thus, the present invention isdirected to polynucleotides having at least a 70% identity, at least an80% identity, at least a 90% identity, or at least a 95% identity to apolynucleotide which encodes the polypeptides set forth in the SequenceListing, as well as fragments thereof, which fragments have at least 30bases or at least 50 bases, and to polypeptides encoded by suchpolynucleotides.

Using the information provided herein, such as the nucleotide sequencesset forth in the Tables and Sequence Listing, nucleic acid molecules ofthe present invention encoding a MGD-CSF polypeptide may be obtainedusing standard cloning and screening procedures, such as those forcloning cDNAs using mRNA as starting material.

Variant and Mutant Polynucleotides

The present invention further relates to variants of the nucleic acidmolecules of the present invention, which encode portions, analogs, orderivatives of the MGD-CSF molecules. Variants may occur naturally, suchas a natural allelic variant, such as one of several alternate forms ofa gene occupying a given locus on a chromosome of an organism, asdescribed in, for example, Genes II, Lewin, B., ed., John Wiley & Sons,New York (1985). Non-naturally occurring variants may be produced usingmutagenesis techniques known in the art.

Such variants include those produced by nucleotide substitutions,deletions, or additions. The substitutions, deletions, or additions mayinvolve one or more nucleotides. The variants may be altered in codingregions, non-coding regions, or both. Alterations in the coding regionsmay produce conservative or non-conservative amino acid substitutions,deletions or additions. These may take the form of silent substitutions,additions, or deletions which do not alter the properties or activitiesof the described MGD-CSF proteins, or portions thereof.

In an embodiment, the invention provides nucleic acid molecules encodingmature proteins, including those with cleaved signal peptide or leadersequences, for example, as shown in the Sequence Listing. Otherembodiments include an isolated nucleic acid molecule comprising apolynucleotide having a nucleotide sequence at least 70% identical, atleast 80% identical, at least 90% identical, or at least 95% identicalto a polynucleotide from the Sequence Listing, a polypeptide encoded bya polynucleotide shown in the Sequence Listing, a polypeptide shown inthe Sequence Listing, or a biologically active fragment of any of these.

A polynucleotide having a nucleotide sequence at least, for example, 95%identical to a reference nucleotide sequence encoding a MGD-CSFpolypeptide is one in which the nucleotide sequence is identical to thereference sequence except that it may include up to five point mutationsper each 100 nucleotides of the reference nucleotide sequence. In otherwords, to obtain a polynucleotide having a nucleotide sequence at least95% identical to a reference nucleotide sequence, up to 5% of thenucleotides in the reference sequence may be deleted or substituted withanother nucleotide, or a number of nucleotides up to 5% of the totalnucleotides in the reference sequence may be inserted into the referencesequence. These mutations of the reference sequence may occur at the 5′or 3′ terminal positions of the reference nucleotide sequence oranywhere between those terminal positions, interspersed eitherindividually among nucleotides in the reference sequence or in one ormore contiguous groups within the reference sequence.

As a practical matter, whether any particular nucleic acid molecule isat least 70%, 80%, 90%, or 95% identical to, for instance, thenucleotide sequences set forth in the Sequence Listing can be determinedconventionally using known computer programs such as the Bestfit program(Wisconsin Sequence Analysis Package, Version 8 for Unix, GeneticsComputer Group, Madison, Wis.). Bestfit uses the local homologyalgorithm of Smith and Waterman, Advances in Applied Mathematics2:482-489 (1981), to find the best segment of homology between twosequences. When using Bestfit or any other sequence alignment program todetermine whether a particular sequence is, for instance, 95% identicalto a reference sequence according to the present invention, theparameters are set, of course, such that the percentage of identity iscalculated over the full length of the reference nucleotide sequence andthat gaps in homology of up to 5% of the total number of nucleotides inthe reference sequence are allowed.

The present application is directed to nucleic acid molecules at least70%, 80%, 90%, or 95% identical to the nucleic acid sequences set forthin the Sequence Listing irrespective of whether they encode apolypeptide having MGD-CSF activity. Even where a particular nucleicacid molecule does not encode a polypeptide having MGD-CSF activity, oneof skill in the art would know how to use the nucleic acid molecule, forinstance, as a hybridization probe or a polymerase chain reaction (PCR)primer. Uses of the nucleic acid molecules of the present invention thatdo not encode a polypeptide having MGD-CSF activity include, inter alia,isolating the MGD-CSF gene or allelic variants thereof in a cDNAlibrary; and in situ hybridization (for example, fluorescent in situhybridization (FISH)) to metaphase chromosomal spreads to provide theprecise chromosomal location of the MGD-CSF genes, as described in Vernaet al., Human Chromosomes: A Manual of Basic Techniques, Pergamon Press,New York (1988); and Northern blot analysis for detecting MGD-CSF mRNAexpression in specific tissues.

The present application is also directed to nucleic acid moleculeshaving sequences at least 70%, 80%, 90%, or 95% identical to a nucleicacid sequence of the Sequence Listing which, encode a polypeptide havingMGD-CSF polypeptide activity, that is, a polypeptide exhibiting activityeither identical to or similar, to an activity of the MGD-CSFpolypeptides of the invention, as measured in a particular biologicalassay. For example, the MGD-CSF polypeptides of the present inventionmay stimulate immune cell proliferation, inhibit tumor growth, and/orkill tumor cells.

Due to the degeneracy of the genetic code, one of ordinary skill in theart will immediately recognize that a large number of the nucleic acidmolecules having a sequence at least 70%, 80%, 90%, or 95% identical tothe nucleic acid sequence of the nucleic acid sequences set forth in theSequence Listing will encode a polypeptide having MGD-CSF polypeptideactivity. In fact, since multiple degenerate variants of thesenucleotide sequences encode the same polypeptide, this will be clear tothe skilled artisan even without performing the above describedcomparison assay. It will be further recognized in the art that areasonable number of nucleic acid molecules that are not degeneratevariants will also encode a polypeptide having MGD-CSF polypeptideactivity, the skilled artisan is fully aware of amino acid substitutionsthat are either less likely or not likely to significantly affectprotein function (for example, replacing one aliphatic amino acid with asecond aliphatic amino acid), as further described below.

Vectors and Host Cells

The present invention also relates to vectors which include the isolatednucleic acid molecules of the present invention, host cells which aregenetically engineered with the recombinant vectors, and the productionof MGD-CSF polypeptides or fragments thereof by recombinant techniques.It provides recombinant vectors that contain, for example, nucleic acidconstructs that encode secretory leader sequences (see, for example, theSequence Listing; the secretory leader may be a collagen secretoryleader), and a selected heterologous polypeptide of interest, and hostcells that are genetically engineered with the recombinant vectors. Thevector may be, for example, a phage, plasmid, or viral vector.Retroviral vectors may be replication competent or replicationdefective. In the latter case, viral propagation generally will occuronly in complementing host cells. Vectors of the invention may containKozak sequences (Lodish et al., Molecular Cell Biology, 4^(th) ed.,1999). Vectors of the invention may also contain the ATG start codon ofthe sequence of interest.

The polynucleotides may be joined to a vector containing a selectablemarker for propagation in a host. Generally, a plasmid vector isintroduced in a precipitate, such as a calcium phosphate precipitate, orin a complex with a charged lipid. If the vector is a virus, it may bepackaged in vitro using an appropriate packaging cell line and thentransduced into host cells.

The DNA insert can be operatively linked to an appropriate promoter,such as the phage lambda PL promoter; the E. coli lac, trp, phoA, andtac promoters; the SV40 early and late promoters; and promoters ofretroviral LTRs, to name a few. Other suitable promoters will be knownto the skilled artisan. The expression constructs will further containsites for transcription initiation, termination, and, in the transcribedregion, a ribosome binding site for translation. The coding portion ofthe transcripts expressed by the constructs can include a translationinitiating codon at the beginning and a termination codon (UAA, UGA, orUAG) appropriately positioned at the end of the polypeptide to betranslated.

The invention provides the expression of genes of interest in animals,including humans, under the control of a promoter that functions, interalfa, in the liver. The hydrodynamics-based procedure of tail veininjection (Zhang et al., Hum. Gene Ther. 10:1735 (1999)) has beendemonstrated to transfect cells with a gene of interest. The inventionalso provides for the manipulation of the level of gene expression bycontrolling the amount and frequency of intravascular DNAadministration. The invention further provides promoters that functionto express genes in the liver.

One large family of proteins expressed in the liver is the cytochromeP450 protein family. These proteins are a group of heme-thiolatemonooxygenases that perform a variety of oxidation reactions, often aspart of the body's mechanism to dispose of harmful substances by makingthem more water-soluble. Much of the body's total mass of cytochromeP450 proteins is found in the liver, specifically, in the microsomes ofhepatocytes. There are over a thousand different cytochrome P450proteins. However, only 49 genes and 15 pseudogenes have been sequencedin humans. In humans, cytochrome P450 3A4 has been identified as themost important cytochrome P450 protein in oxidative metabolism. It isthe most prevalent cytochrome P450 protein in the body, and is aninducible protein.

Operably linking the promoter sequence of genes expressed in the liver,for example the promoter sequence of any of the cytochrome P450 proteinsto a gene of interest can lead to expression of that gene in the liverand any other site where the promoter is active. The inventionencompasses promoters that function to express genes, including, but notlimited to, cytochrome P450 gene, such as cytochrome P450 3A4; c-jun;jun-b; c-fos; c-myc; serum amyloid A; apolipoprotein B editing catalyticsubunit; liver regeneration factors; such as LRF-1 signal transducers,and activators of transcription such as STAT-3; serum alkalinephosphatase (SAP); insulin-like growth factor-binding proteins such asIGFBP-1; cyclin D1; active protein-1; CCAAT enhancer core bindingprotein; beta ornithine decarbonylase; phosphatase of regeneratingliver-1; early growth response gene-1; hepatocyte growth factors;hemopexin; insulin-like growth factors (IGF) such as IGF2; hepatocytenuclear family 1; hepatocyte nuclear family 4; hepatocyte Arg-Ser-richdomain-containing proteins; glucose 6-phosphatase; and acute phaseproteins, such as serum amyloid A and serum amyloid P (SAA/SAP).

As shown in Table 7 and Example 9, operably linking the promotersequence of cytochrome P450 3A4 to MGD-CSF and injecting resulting theconstruct into the tail vein of a mouse induces in the expression ofMGD-CSF and a concomitant increase in monocyte production by the mouse.Thus, the invention provides therapeutic molecules of the invention,delivered in vivo. This method can be used to deliver naked DNA, in thepresence or absence of a pharmaceutically acceptable carrier, or vectorDNA with a sequence of interest. Methods of evaluating the function ofthe molecules of the invention delivered in vivo are known in the art,and some are described herein.

As indicated, the expression vectors may include at least one selectablemarker. Such markers include dihydrofolate reductase, G418 or neomycinresistance for eukaryotic cell culture and tetracycline, kanamycin, orampicillin resistance genes for culturing in E. coli and other bacteria.Representative examples of appropriate hosts include, but are notlimited to, bacterial cells, such as E. coli, Streptomyces, andSalmonella typhimurium cells; fungal cells, such as yeast cells; insectcells such as Drosophila S2 and Spodoptera Sf9 cells; animal cells suchas CHO, COS, 293 and Bowes melanoma cells; and plant cells. Appropriateculture mediums and conditions for the above-described host cells areknown in the art.

The selectable markers are genes that confer a phenotype on a cellexpressing the marker, so that the cell can be identified underappropriate conditions. Generally, a selectable marker allows theselection of transformed cells based on their ability to thrive in thepresence or absence of a chemical or other agent that inhibits anessential cell function. Suitable markers, therefore, include genescoding for proteins which confer drug resistance or sensitivity thereto,impart color to, or change the antigenic characteristics of those cellstransfected with a molecule encoding the selectable marker, when thecells are grown in an appropriate selective medium. For example,selectable markers include cytotoxic markers and drug resistancemarkers, whereby cells are selected by their ability to grow on mediacontaining one or more of the cytotoxins or drugs; auxotrophic markersby which cells are selected for their ability to grow on defined mediawith or without particular nutrients or supplements, such as thymidineand hypoxanthine; metabolic markers for which cells are selected, forexample, their ability to grow on defined media containing theappropriate sugar as the sole carbon source, and markers which conferthe ability of cells to form colored colonies on chromogenic substratesor cause cells to fluoresce.

Among vectors suitable for use in bacteria include pQE70, pQE60, andpQE-9, available from Qiagen, Mississauga, Ontario, Canada; pBS vectors,Phagescript vectors, Bluescript vectors, pNH8A, pNH6a, pNH18A, pNH46A,available from Stratagene (La Jolla, Calif.); and ptrc99a, pKK223-3,pKK233-3, pDR540, pRIT5 available from Pharmacia (Peapack, N.J.). Amongsuitable eukaryotic vectors are pWLNEO, pSV2CAT, pOG44, pXT1, and pSGavailable from Stratagene; and pSVK3, pBPV, pMSG and pSVL, availablefrom Pharmacia. Other suitable vectors will be apparent to the skilledartisan.

Other suitable vectors include those employing a pTT vector backbone(FIG. 2, FIG. 3, and Durocher et al. Nucl. Acids Res. 30 (2002)).Briefly, the pTT vector backbone may be prepared by obtainingpIRESpuro/EGFP (pEGFP) and pSEAP basic vector(s), for example fromClontech (Palo Alto, Calif.), and pcDNA3.1, pcDNA3.1/Myc-(His)₆ (6×Histag disclosed as SEQ ID NO.:277) and pCEP4 vectors can be obtained from,for example, Invitrogen. As used herein, the pTT5 backbone vector cangenerate pTT5-Gateway and be used to transiently express proteins inmammalian cells. The pTT5 vector can be derivatized to pTT5-A, pTT5-B,pTT5-D, pTT5-E, p′175-H, and pTT5-I, for example. As used herein, thepTT2 vector can generate constructs for stable expression in mammaliancell lines.

The expression vector pTT5 allows for extrachromosomal replication ofthe cDNA driven by a cytomegalovirus (CMV) promoter. The plasmid vectorpcDNA-pDEST40 is a Gateway-adapted vector which can utilize a CMVpromoter for high-level expression. SuperGlo GFP variant (sgGFP) can beobtained from Q-Biogene (Carlsbad, Calif.). Preparing a pCEP5 vector canbe accomplished by removing the CMV promoter and polyadenylation signalof pCEP4 by sequential digestion and self-ligation using SalI and XbaIenzymes resulting in plasmid pCEP4A. A GblII fragment from pAdCMV5(Massie et al., J. Virol., 72: 2289-2296 (1998)), encoding theCMV5-poly(A) expression cassette ligated in BglII-linearized pCEP4A,resulting in pCEP5 vector.

The pTT vector can be prepared by deleting the hygromycin (BsmI and SalIexcision followed by fill-in and ligation) and EBNA1 (ClaI and NsiIexcision followed by fill-in and ligation) expression cassettes. TheColEI origin (FspI-SalI fragment, including the 3′ end of β-lactamaseORF) can be replaced with a FspI-SalI fragment from pcDNA3.1 containingthe pMBI origin (and the same 3′ end of β-lactamase ORF). A Myc-(His)₆(6×His tag disclosed as SEQ ID NO.:277) C-terminal fusion tag can beadded to SEAP (HindIII-HpaI fragment from pSEAP-basic) followingin-frame ligation in pcDNA3.1/Myc-His digested with HindIII and EcoRV.Plasmids can subsequently be amplified in E. coli (DH5a) grown in LBmedium and purified using MAXI prep columns (Qiagen, Mississauga,Ontario, Canada). To quantify, plasmids can be subsequently diluted in50 mM Tris-HCl pH 7.4 and absorbencies can be measured at 260 nm and 280nm. Plasmid preparations with A₂₆₀/A₂₈₀ ratios between about 1.75 andabout 2.00 are suitable.

Introduction of the construct into the host cell can be effected bycalcium phosphate transfection, DEAE-dextran mediated transfection,cationic lipid-mediated transfection, electroporation, transduction,infection, or other methods. Such methods are described in many standardlaboratory manuals, such as Sambrook, J., et al. (2001) MolecularCloning, A Laboratory Manual. 3^(rd) ed. Cold Spring Harbor LaboratoryPress.

The polypeptides may be expressed in a modified form, such as a fusionprotein, and may include not only secretion signals, but also additionalheterologous functional regions. For instance, a region of additionalamino acids, particularly charged amino acids, may be added to theN-terminus of the polypeptide to improve stability and persistence inthe host cell, during purification, or during subsequent handling andstorage. Also, peptide moieties may be added to the polypeptide tofacilitate purification. Such regions may be removed prior to finalpreparation of the polypeptide.

Polypeptides

The invention further provides isolated MGD-CSF polypeptides containingthe amino acid sequences encoded by the nucleotide sequences set forthin the Tables and Sequence

Listing for example, SEQ. ID. NOS.:7, 8, 9, and 11, which correspond tothe full-length polypeptide, exon 4, the mature polypeptide, and thefragment TRLRAQ (SEQ ID NO.:11) (present at the junction between exon 3and exon 4 of MGD-CSF), respectively. The invention provides novel usesfor these novel polypeptides and for related polypeptides, such as thoseshown in SEQ. ID. NOS.:10 and 12.

The invention provides secreted proteins, which are capable of beingdirected to the ER, secretory vesicles, or the extracellular space as aresult of a secretory leader, signal peptide, or leader sequence, aswell as proteins released into the extracellular space withoutnecessarily containing a signal sequence. If a secreted protein isreleased into the extracellular space, it may undergo extracellularprocessing to a mature polypeptide. Release into the extracellular spacecan occur by many mechanisms, including exocytosis and proteolyticcleavage.

As shown in FIG. 8A, the MGD-CSF polypeptides can be recovered andisolated from recombinant cell cultures by well-known methods. Suchmethods include ammonium sulfate or ethanol precipitation, acidextraction, anion or cation exchange chromatography, phosphocellulosechromatography, hydrophobic interaction chromatography, affinitychromatography, hydroxylapatite chromatography, and lectinchromatography. High performance liquid chromatography (HPLC) can beemployed for purification. Polypeptides of the present invention includeproducts purified from natural sources, including bodily fluids, tissuesand cells, whether directly isolated or cultured; products of chemicalsynthetic procedures; and products produced by recombinant techniquesfrom a prokaryotic or eukaryotic host, including, for example,bacterial, yeast, higher plant, insect, and mammalian cells. Dependingupon the host employed in a recombinant production procedure, thepolypeptides of the present invention may be glycosylated or may benon-glycosylated. In addition, polypeptides of the invention may alsoinclude an initial modified methionine residue, in some cases as aresult of host-mediated processes. Thus, it is well known in the artthat the N-terminal methionine encoded by the translation initiationcodon generally is removed with high efficiency from any protein aftertranslation in eukaryotic cells. While the N-terminal methionine on mostproteins also is efficiently removed in most prokaryotes, for someproteins this prokaryotic removal process is inefficient, depending onthe nature of the amino acid to which the N-terminal methionine iscovalently linked.

Typically, a heterologous polypeptide, whether modified or unmodified,may be expressed as described above, or as a fusion protein, and mayinclude not only secretion signals, but also a secretory leadersequence. A secretory leader sequence of the invention directs certainproteins to the endoplasmic reticulum (ER). The ER separates themembrane-bound proteins from other proteins. Once localized to the ER,proteins can be further directed to the Golgi apparatus for distributionto vesicles, including secretory vesicles; the plasma membrane;lysosomes; and other organelles.

Proteins targeted to the ER by a secretory leader sequence can bereleased into the extracellular space as a secreted protein. Forexample, vesicles containing secreted proteins can fuse with the cellmembrane and release their contents into the extracellular space viaexocytosis. Exocytosis can occur constitutively or upon receipt of atriggering signal. In the latter case, the proteins may be stored insecretory vesicles (or secretory granules) until exocytosis istriggered. Similarly, proteins residing on the cell membrane can also besecreted into the extracellular space by proteolytic cleavage of alinker holding the protein to the membrane.

Additionally, peptide moieties and/or purification tags may be added tothe polypeptide to facilitate purification. Such regions may be removedprior to final preparation of the polypeptide. The addition of peptidemoieties to polypeptides to engender secretion or excretion, to improvestability, and to facilitate purification, among other reasons, arefamiliar and routine techniques in the art. Suitable purification tagsinclude, for example, V5, HISX6 (SEQ ID NO.:277), HISX8 (SEQ IDNO.:278), avidin, and biotin.

The invention provides a fusion protein comprising a heterologous regionfrom an immunoglobulin that is useful to stabilize and purify proteins.The addition of peptide moieties to polypeptides to engender secretionor excretion, to improve stability and to facilitate purification, amongothers, are familiar and routine techniques in the art. For example, EP0 464 533 (Canadian counterpart 2045869) discloses fusion proteinscontaining various portions of constant region of immunoglobulinmolecules together with another human protein or part thereof. In manycases, the Fc part of a fusion protein is advantageous for use intherapy and diagnosis and thus results, for example, in improvedpharmacokinetic properties (EP 0 232 262). On the other hand, for someuses it would be desirable to be able to delete the Fc part after thefusion protein has been expressed, detected, and purified in theadvantageous manner described. This is the case when the Fc portionproves to be a hindrance to use in therapy and/or diagnosis, forexample, when the fusion protein is to be used as an antigen forimmunizations. In drug discovery, for example, human proteins, such ashIL-5, have been fused with Fc portions for the purpose ofhigh-throughput screening assays to identify antagonists of hIL-5. See,Bennett et al., J. Molec. Recog., 8:52-58 (1995) and Johanson et al, J.Biol. Chem., 270:9459-9471 (1995).

The polypeptides of the present invention can be provided in an isolatedform, and can be substantially purified, as described above. Arecombinantly produced version of the herein described MGD-CSFpolypeptides can also be substantially isolated, for example, accordingto the one-step method described in Smith and Johnson, Gene, 67:31-40(1988). Polypeptides of the invention can further be isolated fromnatural or recombinant sources using anti-MGD-CSF antibodies of theinvention produced using methods well known in the art.

The polypeptides herein may be purified or isolated in the presence ofions or agents that aid in the refolding of the molecules or aid indimerizing or trimerizing the molecules as conventional in the art. Forexample, cofactors may be added to promote physiologic folding ormultimerization.

Further polypeptides of the present invention include polypeptides whichhave at least 70%, 80%, 90%, or 95% identity to those described above.The polypeptides of the invention also contain those which are at least70%, 80%, 90%, or 95% identical to a polypeptide encoded by a nucleicacid sequence of the Sequence Listing.

The % identity of two polypeptides can be measured by a similarity scoredetermined by comparing the amino acid sequences of the two polypeptidesusing the Bestfit program with the default settings for determiningsimilarity. Bestfit uses the local homology algorithm of Smith andWaterman, Advances in Applied Mathematics 2:482-489 (1981) to find thebest segment of similarity between two sequences.

A polypeptide having an amino acid sequence at least, for example, 95%identical to a reference amino acid sequence of a MGD-CSF polypeptide isone in which the amino acid sequence of the polypeptide is identical tothe reference sequence except that the polypeptide sequence may includeup to five amino acid alterations per each 100 amino acids of thereference polypeptide. In other words, to obtain a polypeptide having anamino acid sequence at least 95% identical to a reference amino acidsequence, up to 5% of the amino acid residues in the reference sequencemay be deleted or substituted with another amino acid, or a number ofamino acids, up to 5% of the total amino acid residues in the referencesequence, may be inserted into the reference sequence. These alterationsof the reference sequence may occur at the amino or carboxy terminalpositions of the reference amino acid sequence or anywhere between thoseterminal positions, interspersed either individually among residues inthe reference sequence, or in one or more contiguous groups within thereference sequence.

As a practical matter, whether any particular polypeptide is at least70%, 80%, 90%, or 95% identical to, for instance, an amino acid sequenceor to a polypeptide sequence encoded by a nucleic acid sequence setforth in the Sequence Listing can be determined conventionally usingknown computer programs, such the Bestfit program. When using Bestfit orother sequence alignment program to determine whether a particularsequence is, for instance, 95% identical to a reference sequenceaccording to the present invention, the parameters are set, of course,that the percentage of identity is calculated over the full length ofthe reference amino acid sequence and that gaps in homology of up to 5%of the total number of amino acid residues in the reference sequence areallowed.

Variant and Mutant Polypeptides

Protein engineering may be employed to improve or alter thecharacteristics of MGD-CSF polypeptides of the invention. RecombinantDNA technology known to those skilled in the art can be used to createnovel mutant proteins or “muteins” including single or multiple aminoacid substitutions, deletions, additions, or fusion proteins. Suchmodified polypeptides can show desirable properties, such as enhancedactivity or increased stability. In addition, they may be purified inhigher yields and show better solubility than the corresponding naturalpolypeptide, at least under certain purification and storage conditions.

N-Terminal and C-Terminal Deletion Mutants

For instance, for many proteins, including the extracellular domain of amembrane associated protein or the mature form(s) of a secreted protein,it is known in the art that one or more amino acids may be deleted fromthe N-terminus or C-terminus without substantial loss of biologicalfunction. For instance, Ron et al., J. Biol. Chem., 268:2984-2988(1993), reported modified KGF proteins that had heparin binding activityeven if 3, 8, or 27 amino-terminal amino acid residues were missing.

However, even if deletion of one or more amino acids from the N-terminusof a protein results in modification or loss of one or more biologicalfunctions of the protein, other biological activities may still beretained. Thus, the ability of the shortened protein to induce and/orbind to antibodies which recognize the complete or mature from of theprotein generally will be retained when less than the majority of theresidues of the complete or mature protein are removed from theN-terminus. Whether a particular polypeptide lacking N-terminal residuesof a complete protein retains such immunologic activities can bedetermined by routine methods described herein and otherwise known inthe art. Accordingly, the present invention further providespolypeptides having one or more residues deleted from the amino terminusof the amino acid sequences of the MGD-CSF molecules as shown in theSequence Listing.

Similarly, many examples of biologically functional C-terminal deletionmuteins are known. For instance, interferon gamma increases in activityas much as ten fold when 8-10 amino acid residues are deleted from thecarboxy terminus of the protein, see, for example, Dobeli et al., J.Biotechnology, 7:199-216 (1988).

However, even if deletion of one or more amino acids from the C-terminusof a protein results in modification of loss of one or more biologicalfunctions of the protein, other biological activities may still beretained. Thus, the ability of the shortened protein to induce and/orbind to antibodies which recognize the complete or mature form of theprotein generally will be retained when less than the majority of theresidues of the complete or mature protein are removed from theC-terminus. Whether a particular polypeptide lacking C-terminal residuesof a complete protein retains such immunologic activities can bedetermined by routine methods described herein and otherwise known inthe art.

Cysteine to Serine Muteins

The MGD-CSF sequence includes seven cysteine residues, located at aminoacid positions 35, 167, 176, 178, 179, 190, and 198. In an embodiment,the invention provides mutant MGC34647 molecules with serine mutated tocysteine. These mutants are shown in Table 1 and in the SequenceListing, designated SEQ. ID. NOS.:258-271. The collagen signal peptidedescribed by SEQ. ID. NO.:14 was used to improve the secretion of theexpressed polypeptide. As set forth in Table 1 and FIG. 8B and explainedin more detail in Example 18, the cysteines at positions 35, 167, 176,178, 179, 190, and 198 were each substituted for serine. Theseconstructs may be cloned into any suitable vector, as known in the art,for example, the pTT5-G vector.

Analyzing these muteins provides an understanding of the disulfide bondpattern of MGD-CSF and may identify a protein with improved properties,for example, improved expression and secretion from mammalian cells,decreased aggregation of the purified protein, and the potential toproduce active recombinant MGD-CSF, when expressed in E. coli.

Other Mutants

In addition to terminal deletion forms of the protein discussed above,it also will be recognized by one of ordinary skill in the art that someamino acid sequences of the MGD-CSF polypeptides can be varied withoutsignificant effect of the structure or function of the protein. If suchdifferences in sequence are contemplated, it should be remembered thatthere will be critical areas on the protein which determine activity.

Thus, the invention further includes variations of the MGD-CSFpolypeptides which show substantial MGD-CSF polypeptide activity orwhich include regions of the MGD-CSF proteins such as the proteinportions discussed below. Such mutants include deletions, insertions,inversions, repeats, and type substitutions, selected according togeneral rules known in the art, so as have little effect on activity.For example, guidance concerning how to make phenotypically silent aminoacid substitutions is provided in Bowie et al., Science, 247:1306-1310(1990), wherein the authors indicate that there are two main approachesfor studying the tolerance of an amino acid sequence to change. Thefirst method relies on the process of evolution, in which mutations areeither accepted or rejected by natural selection. The second approachuses genetic engineering to introduce amino acid changes at specificpositions of a cloned gene and selections, or screens, to identifysequences that maintain functionality.

These studies report that proteins are surprisingly tolerant of aminoacid substitutions. The authors further indicate which amino acidchanges are likely to be permissive at a certain position of theprotein. For example, most buried amino acid residues require nonpolarside chains, whereas few features of surface side chains are generallyconserved. Other such phenotypically silent substitutions are describedin Bowie, et al., supra, and the references cited therein. Typicallyseen as conservative substitutions are the replacements, one foranother, among the aliphatic amino acids Ala, Val, Leu, and Ile;interchange of the hydroxyl residues Ser and Thr, exchange of the acidicresidues Asp and Glu, substitution between the amide residues Asn andGln, exchange of the basic residues Lys and Arg, and replacementsbetween the aromatic residues Phe and Tyr.

Thus, a fragment, derivative, or analog of a polypeptide of the SequenceListing or polypeptide encoded by a nucleic acid sequence of theSequence Listing may be (i) one in which one or more of the amino acidresidues are substituted with a conserved or non-conserved amino acidresidue; such a substituted amino acid residue may or may not be oneencoded by the genetic code; (ii) one in which one or more of the aminoacid residues includes a substituent group; (iii) one in which themature polypeptide is fused with another compound, such as a compound toincrease the half-life of the polypeptide (for example, polyethyleneglycol); or (iv) one in which the additional amino acids are fused tothe above form of the polypeptide, such as an IgG Fc fusion regionpeptide, a leader or secretory sequence, a sequence employed to purifythe above form of the polypeptide, or a proprotein sequence. Suchfragments, derivatives, and analogs are deemed to be within the scope ofthose skilled in the art from the teachings herein.

Thus, the MGD-CSF polypeptides of the present invention may include oneor more amino acid substitutions, deletions, or additions, either fromnatural mutations or human manipulation. As indicated, these changes maybe of a minor nature, such as conservative amino acid substitutions,that do not significantly affect the folding or activity of the protein.Conservative amino acid substitutions include the aromatic substitutionsPhe, Trp, and Tyr; the hydrophobic substitutions Leu, Iso, and Val; thepolar substitutions Glu and Asp; the basic substitutions Arg, Lys, andHis; the acidic substitutions Asp and Glu; and the small amino acidsubstations Ala, Ser, Thr, Met, and Gly.

Amino acids essential for the functions of MGD-CSF polypeptides can beidentified by methods known in the art, such as site-directedmutagenesis or alanine-scanning mutagenesis, see, for example,Cunningham and Wells, Science, 244:1081-1085 (1989). The latterprocedure introduces single alanine mutations. The resulting mutantmolecules are then tested for biological activity such as receptorbinding, or in vitro or in vitro proliferative activity.

Of special interest are substitutions of charged amino acids with othercharged or neutral amino acids which may produce proteins with highlydesirable improved characteristics, such as less aggregation.Aggregation may not only reduce activity but also be problematic whenpreparing pharmaceutical formulations, because, for example, aggregatescan be immunogenic, Pinckard et al., Clin. Exp. Immunol., 2:331-340(1967); Robbins et al., Diabetes, 36:838-845 (1987); Cleland et al.,Crit. Rev. Therapeutic Drug Carrier Systems, 10:307-377 (1993).

Replacing amino acids can also change the selectivity of the binding ofa ligand to cell surface receptors. For example, Ostade et al., Nature,361:266-268 (1993) describes mutations resulting in selective binding ofTNF-α to only one of the two known types of TNF receptors. Sites thatare critical for ligand-receptor binding can also be determined bystructural analysis such as crystallization, nuclear magnetic resonance,or photoaffinity labeling, for example, Smith et al., J. Mol. Biol.,224:899-904 (1992) and de Vos et al., Science, 255:306-312 (1992).

Epitope-Bearing Portions

As described in detail below, the polypeptides of the present inventioncan be used to raise polyclonal and monoclonal antibodies, which areuseful in assays for detecting MGD-CSF protein expression, also asdescribed below, or as agonists and/or antagonists capable of enhancingor inhibiting MGD-CSF protein function. These polypeptides can also beused in a yeast two-hybrid system to capture MGD-CSF protein bindingproteins, which are also candidate agonists and antagonists, accordingto the present invention. The yeast two hybrid system is described inFields and Song, Nature, 340:245-246 (1989).

In another aspect, the invention provides a polypeptide comprising oneor more epitope-bearing portion of a polypeptide of the invention. Theinvention provides polyclonal antibodies specific to MGD-CSF andprovides that MGD-CSF has, at minimum, two antigenic epitopes. Theepitope of this polypeptide portion is an immunogenic or antigenicepitope of a polypeptide of the invention. Immunogenic epitopes arethose parts of a protein that elicit an antibody response when the wholeprotein is provided as the immunogen. On the other hand, a region of aprotein molecule to which an antibody can bind is an antigenic epitope.The number of immunogenic epitopes of a protein generally is less thanthe number of antigenic epitopes. See, for instance, Geysen et al.,Proc. Natl. Acad. Sci., 81:3998-4002 (1983).

As to the selection of polypeptides bearing an antigenic epitope (thatis, those which contain a region of a protein molecule to which anantibody can bind), it is well known in that art that relatively shortsynthetic peptides that mimic part of a protein sequence are routinelycapable of eliciting an antiserum that reacts with the partiallymimicked protein. See, for instance, Sutcliffe et al., Science,219:660-666 (1983). Peptides capable of eliciting protein-reactive seraare frequently represented in the primary sequence of a protein, can becharacterized by a set of simple chemical rules, and are confinedneither to immunodominant regions of intact proteins (that is, toimmunogenic epitopes) nor to the amino or carboxyl terminals. Antigenicepitope-bearing peptides and polypeptides of the invention are thereforeuseful for raising antibodies, including monoclonal antibodies, thatbind specifically to a polypeptide of the invention. See, for instance,Wilson et al., Cell, 37:767-778 (1984). The epitope-bearing peptides andpolypeptides of the invention may be produced by any conventional means.See, for example, Houghten, Proc. Natl. Acad. Sci. 82:5131-5135 (1985),and U.S. Pat. No. 4,631,211 (1986).

Epitope-bearing peptides and polypeptides of the invention can be usedto induce antibodies according to methods well known in the art. See,for instance, Bittle, et al, J. Gen. Virol., 66:2347-2354 (1985).Immunogenic epitope-bearing peptides of the invention, those parts of aprotein that elicit an antibody response when the whole protein is theimmunogen, are identified according to methods known in the art. See,for instance, U.S. Pat. No. 5,194,392 (1990), which describes a generalmethod of detecting or determining the sequence of monomers (amino acidsor other compounds) which is a topological equivalent of the epitope(mimotope) which is complementary to a particular antigen binding site(paratope) of an antibody of interest. More generally, U.S. Pat. No.4,433,092 (1989) describes a method of detecting or determining asequence of monomers which is a topographical equivalent of a ligandwhich is complementary to the ligand binding site of a particularreceptor of interest. Similarly, U.S. Pat. No. 5,480,971 (1996)discloses linear C1-C7-alkyl peralkylated oligopeptides, and sets andlibraries of such peptides, as well as methods for using sucholigopeptide sets and libraries for determining the sequence of aperalkylated oligopeptide that, for example, binds to an acceptormolecule of interest. Thus, non-peptide analogs of the epitope-bearingpeptides of the invention also can be made routinely by these methods.

Fusion Molecules

As one of skill in the art will appreciate, MGD-CSF polypeptides of thepresent invention, and the epitope-bearing fragments thereof describedabove, can be combined with heterologous polypeptides, resulting inchimeric polypeptides. These fusion proteins facilitate purification andshow an increased half-life in vivo. This has been reported, forexample, in chimeric proteins consisting of the first two domains of thehuman CD4-polypeptide and various domains of the constant regions of theheavy or light chains of mammalian immunoglobulins, for example, EP 0394 827; Traunecker et al., Nature, 331:84-86 (1988). Fusion proteinsthat have a disulfide-linked dimeric structure due to the IgG portioncan also be more efficient in binding and neutralizing other moleculesthan the monomeric MGD-CSF protein or protein fragment alone, forexample, as described by Fountoulakis et al., J. Biochem., 270:3958-3964(1995). Suitable chemical moieties for derivatization of a heterologouspolypeptide include, for example, polymers, such as water solublepolymers, the constant domain of immunoglobulins, all or part of humanserum albumin; fetuin A; fetuin B; a leucine zipper domain; atetranectin trimerization domain; mannose binding protein (also known asmannose binding lectin), for example, mannose binding protein 1; and anFc region, as described herein and further described in U.S. Pat. No.6,686,179, and U.S. Application Nos. 60/589,788 and 60/654,229. Methodsof making fusion proteins are well-known to the skilled artisan.

For example, the short plasma half-life of unmodified interferon alphamakes frequent dosing necessary over an extended period of time, inorder to treat viral and proliferative disorders. Interferon alpha fusedwith HSA has a longer half life and requires less frequent dosing thanunmodified interferon alpha; the half-life was 18-fold longer and theclearance rate was approximately 140 times slower (Osborn et al., J.Pharmacol. Exp. Ther. 303:540-548, 2002). Interferon beta fused with HSAalso has favorable pharmacokinetic properties; its half life wasreported to be 36-40 hours, compared to 8 hours for unmodifiedinterferon beta (Sung et al., J. Interferon Cytokine Res. 23:25-36,2003). A HSA-interleukin-2 fusion protein has been reported to have botha longer half-life and favorable biodistribution compared to unmodifiedinterleukin-2. This fusion protein was observed to target tissues wherelymphocytes reside to a greater extent than unmodified interleukin 2,suggesting that it exerts greater efficacy (Yao et al., Cancer Immunol.Immunother. 53:404-410, 2004).

The Fc receptor of human immunoglobulin G subclass 1 has also been usedas a fusion partner for a therapeutic molecule. It has beenrecombinantly linked to two soluble p75 tumor necrosis factor (TNF)receptor molecules. This fusion protein has been reported to have alonger circulating half-life than monomeric soluble receptors, and toinhibit TNFα-induced proinflammatory activity in the joints of patientswith rheumatoid arthritis (Goldenberg, Clin. Ther. 21:75-87, 1999). Thisfusion protein has been used clinically to treat rheumatoid arthritis,juvenile rheumatoid arthritis, psoriatic arthritis, and ankylosingspondylitis (Nanda and Bathon, Expert Opin. Pharmacother. 5:1175-1186,2004).

Polymers, for example, water soluble polymers, are useful in the presentinvention as the polypeptide to which each polymer is attached will notprecipitate in an aqueous environment, such as typically found in aphysiological environment. Polymers employed in the invention will bepharmaceutically acceptable for the preparation of a therapeutic productor composition. One skilled in the art will be able to select thedesired polymer based on such considerations as whether thepolymer/protein conjugate will be used therapeutically and, if so, thedesired dosage, circulation time, and resistance to proteolysis.

Suitable, clinically acceptable, water soluble polymers include, but arenot limited to, polyethylene glycol (PEG), polyethylene glycolpropionaldehyde, copolymers of ethylene glycol/propylene glycol,monomethoxy-polyethylene glycol, carboxymethyl cellulose, dextran,polyvinyl alcohol (PVA), polyvinyl pyrrolidone, poly-1,3-dioxolane,poly-1,3,6-trioxane, ethylene/maleic anhydride copolymer, poly (β-aminoacids) (either homopolymers or random copolymers), poly(n-vinylpyrrolidone) polyethylene glycol, polypropylene glycol homopolymers(PPG) and other polyakylene oxides, polypropylene oxide/ethylene oxidecopolymers, polyoxyethylated polyols (POG) (e.g., glycerol) and otherpolyoxyethylated polyols, polyoxyethylated sorbitol, or polyoxyethylatedglucose, colonic acids or other carbohydrate polymers, Ficoll, ordextran and mixtures thereof.

As used herein, polyethylene glycol (PEG) is meant to encompass any ofthe forms that have been used to derivatize other proteins, such asmono-(C1-C10) alkoxy- or aryloxy-polyethylene glycol. Polyethyleneglycol propionaldehyde may have advantages in manufacturing due to itsstability in water.

Specifically, a modified heterologous polypeptide of the invention maybe prepared by attaching polyaminoacids or branch point amino acids tothe polypeptide. For example, the polyaminoacid may be a carrier proteinthat serves to increase the circulation half life of the polypeptide (inaddition to the advantages achieved via a fusion molecule). For thetherapeutic purpose of the present invention, such polyaminoacids shouldideally be those that have or do not create neutralizing antigenicresponse, or other adverse responses. Such polyaminoacids may be chosenfrom serum album (such as human serum albumin), an additional antibodyor portion thereof, for example the Fc region, fetuin A, fetuin B,leucine zipper nuclear factor erythroid derivative-2 (NFE2),neuroretinal leucine zipper, tetranectin, or other polyaminoacids, forexample, lysines. As described herein, the location of attachment of thepolyaminoacid may be at the N-terminus, or C-terminus, or other placesin between, and also may be connected by a chemical linker moiety to theselected molecule.

Polymers used herein, for example water soluble polymers, may be of anymolecular weight and may be branched or unbranched. The polymers eachtypically have an average molecular weight of between about 2 kDa toabout 100 kDa (the term “about” indicating that in preparations of apolymer, some molecules will weigh more, some less, than the statedmolecular weight). The average molecular weight of each polymer may bebetween about 5 kDa and about 50 kDa, or between about 12 kDa and about25 kDa. Generally, the higher the molecular weight or the more branches,the higher the polymer:protein ratio. Other sizes may also be used,depending on the desired therapeutic profile; for example, the durationof sustained release; the effects, if any, on biological activity; theease in handling; the degree or lack of antigenicity; and other knowneffects of a polymer on a modified molecule of the invention.

Polymers employed in the present invention are typically attached to aheterologous polypeptide with consideration of effects on functional orantigenic domains of the polypeptide. In general, chemicalderivatization may be performed under any suitable condition used toreact a protein with an activated polymer molecule. Activating groupswhich can be used to link the polymer to the active moieties includesulfone, maleimide, sulfhydryl, thiol, triflate, tresylate, azidirine,oxirane, and 5-pyridyl.

Polymers of the invention are typically attached to a heterologouspolypeptide at the alpha (α) or epsilon (ε) amino groups of amino acidsor a reactive thiol group, but it is also contemplated that a polymergroup could be attached to any reactive group of the protein that issufficiently reactive to become attached to a polymer group undersuitable reaction conditions. Thus, a polymer may be covalently bound toa heterologous polypeptide via a reactive group, such as a free amino orcarboxyl group. The amino acid residues having a free amino group mayinclude lysine residues and the N-terminal amino acid residue. Thosehaving a free carboxyl group may include aspartic acid residues,glutamic acid residues, and the C-terminal amino acid residue. Thosehaving a reactive thiol group include cysteine residues.

Methods for preparing fusion molecules conjugated with polymers, such aswater soluble polymers, will each generally involve (a) reacting aheterologous polypeptide with a polymer under conditions whereby thepolypeptide becomes attached to one or more polymers and (b) obtainingthe reaction product. Reaction conditions for each conjugation may beselected from any of those known in the art or those subsequentlydeveloped, but should be selected to avoid or limit exposure to reactionconditions such as temperatures, solvents, and pH levels that wouldinactivate the protein to be modified. In general, the optimal reactionconditions for the reactions will be determined case-by-case based onknown parameters and the desired result. For example, the larger theratio of polymer:polypeptide conjugate, the greater the percentage ofconjugated product. The optimum ratio (in terms of efficiency ofreaction in that there is no excess unreacted polypeptide or polymer)may be determined by factors such as the desired degree ofderivatization (e.g., mono-, di-tri- etc.), the molecular weight of thepolymer selected, whether the polymer is branched or unbranched and thereaction conditions used. The ratio of polymer (for example, PEG) to apolypeptide will generally range from 1:1 to 100:1. One or more purifiedconjugates may be prepared from each mixture by standard purificationtechniques, including among others, dialysis, salting-out,ultrafiltration, ion-exchange chromatography, gel filtrationchromatography, and electrophoresis.

One may specifically desire an N-terminal chemically modified protein.One may select a polymer by molecular weight, branching, etc., theproportion of polymers to protein (polypeptide or peptide) molecules inthe reaction mix, the type of reaction to be performed, and the methodof obtaining the selected N-terminal chemically modified protein. Themethod of obtaining the N-terminal chemically modified proteinpreparation (separating this moiety from other monoderivatized moietiesif necessary) may be by purification of the N-terminal chemicallymodified protein material from a population of chemically modifiedprotein molecules.

Selective N-terminal chemical modification may be accomplished byreductive alkylation which exploits differential reactivity of differenttypes of primary amino groups (lysine versus the N-terminal) availablefor derivatization in a particular protein. Under the appropriatereaction conditions, substantially selective derivatization of theprotein at the N-terminus with a carbonyl group containing polymer isachieved. For example, one may selectively attach a polymer to theN-terminus of the protein by performing the reaction at a pH whichallows one to take advantage of the pKa differences between the 6-aminogroup of the lysine residues and that of the α-amino group of theN-terminal residue of the protein. By such selective derivatization,attachment of a polymer to a protein is controlled: the conjugation withthe polymer takes place predominantly at the N-terminus of the proteinand no significant modification of other reactive groups, such as thelysine side chain amino groups, occurs. Using reductive alkylation, thepolymer may be of the type described above and should have a singlereactive aldehyde for coupling to the protein. Polyethylene glycolpropionaldehyde, containing a single reactive aldehyde, may also beused.

In one embodiment, the present invention contemplates the chemicallyderivatized polypeptide to include mono- or poly- (e.g., 2-4) PEGmoieties. Pegylation may be carried out by any of the pegylationreactions known in the art. Methods for preparing a pegylated proteinproduct will generally include (a) reacting a polypeptide withpolyethylene glycol (such as a reactive ester or aldehyde derivative ofPEG) under conditions whereby the protein becomes attached to one ormore PEG groups; and (b) obtaining the reaction product(s). In general,the optimal reaction conditions for the reactions will be determinedcase by case based on known parameters and the desired result.

There are a number of PEG attachment methods available to those skilledin the art. See, for example, EP 0 401 384; Malik et al., Exp. Hematol.,20:1028-1035 (1992); Francis, Focus on Growth Factors, 3(2):4-10 (1992);EP 0 154 316; EP 0 401 384; WO 92/16221; WO 95/34326; and the otherpublications cited herein that relate to pegylation.

The step of pegylation as described herein may be carried out via anacylation reaction or an alkylation reaction with a reactivepolyethylene glycol molecule. Thus, protein products according to thepresent invention include pegylated proteins wherein the PEG group(s) is(are) attached via acyl or alkyl groups. Such products may bemono-pegylated or poly-pegylated (for example, those containing 2-6 or2-5 PEG groups). The PEG groups are generally attached to the protein atthe α- or ε-amino groups of amino acids, but it is also contemplatedthat the PEG groups could be attached to any amino group attached to theprotein that is sufficiently reactive to become attached to a PEG groupunder suitable reaction conditions.

Pegylation by acylation generally involves reacting an active esterderivative of polyethylene glycol (PEG) with a polypeptide of theinvention. For acylation reactions, the polymer(s) selected typicallyhave a single reactive ester group. Any known or subsequently discoveredreactive PEG molecule may be used to carry out the pegylation reaction.An example of a suitable activated PEG ester is PEG esterified toN-hydroxysuccinimide (NHS). As used herein, acylation is contemplated toinclude, without limitation, the following types of linkages between thetherapeutic protein and a polymer such as PEG: amide, carbamate,urethane, and the like, see for example, Chamow, Bioconjugate Chem.,5:133-140 (1994). Reaction conditions may be selected from any of thoseknown in the pegylation art or those subsequently developed, but shouldavoid conditions such as temperature, solvent, and pH that wouldinactivate the polypeptide to be modified.

Pegylation by acylation will generally result in a poly-pegylatedprotein. The connecting linkage may be an amide. The resulting productmay be substantially only (e.g., >95%) mono, di- or tri-pegylated.However, some species with higher degrees of pegylation may be formed inamounts depending on the specific reaction conditions used. If desired,more purified pegylated species may be separated from the mixture(particularly unreacted species) by standard purification techniques,including among others, dialysis, salting-out, ultrafiltration,ion-exchange chromatography, gel filtration chromatography andelectrophoresis.

Pegylation by alkylation generally involves reacting a terminal aldehydederivative of PEG with a polypeptide in the presence of a reducingagent. For the reductive alkylation reaction, the polymer(s) selectedshould have a single reactive aldehyde group. An exemplary reactive PEGaldehyde is polyethylene glycol propionaldehyde, which is water stable,or mono C1-C10 alkoxy or aryloxy derivatives thereof, see for example,U.S. Pat. No. 5,252,714.

Additionally, heterologous polypeptides of the present invention and theepitope-bearing fragments thereof described herein can be combined withparts of the constant domain of immunoglobulins (IgG), resulting inchimeric polypeptides. These particular fusion molecules facilitatepurification and show an increased half-life in vivo. This has beenshown, for example, in chimeric proteins consisting of the first twodomains of the human CD4-polypeptide and various domains of the constantregions of the heavy or light chains of mammalian immunoglobulins, suchas EP 0 394 827; Traunecker et al., Nature, 331:84-86 (1988). Fusionmolecules that have a disulfide-linked dimeric structure due to the IgGpart can also be more efficient in binding and neutralizing othermolecules than, for example, a monomeric polypeptide or polypeptidefragment alone; see, for example, Fountoulakis et al., J. Biochem.,270:3958-3964 (1995).

In another described embodiment, a human serum albumin fusion moleculemay also be prepared as described herein and as further described inU.S. Pat. No. 6,686,179.

Moreover, the polypeptides of the present invention can be fused tomarker sequences, such as a peptide that facilitates purification of thefused polypeptide. The marker amino acid sequence may be ahexa-histidine peptide such as the tag provided in a pQE vector (Qiagen,Mississauga, Ontario, Canada), among others, many of which arecommercially available. As described in Gentz et al., Proc. Natl. Acad.Sci. 86:821-824 (1989), for instance, hexa-histidine provides forconvenient purification of the fusion protein. Another peptide taguseful for purification, the hemagglutinin HA tag, corresponds to anepitope derived from the influenza hemagglutinin protein. (Wilson etal., Cell 37:767 (1984)). Any of these above fusions can be engineeredusing the polynucleotides or the polypeptides of the present invention.

Secretory Leader Sequences

As demonstrated herein, and in U.S. 60/647,013, in order for somesecreted proteins to express and secrete in larger quantities, asecretory leader sequence from another, different, secreted protein isdesirable. Employing heterologous secretory leader sequences isadvantageous in that a resulting mature amino acid sequence of thesecreted polypeptide is not altered as the secretory leader sequence isremoved in the ER during the secretion process. Moreover, the additionof a heterologous secretory leader is required to express and secretesome proteins.

Thus, to identify potential robust secretory leader sequence(s) thatcould universally be used to secrete proteins and to express MGD-CSF,Applicants have cloned and expressed a number of different secretedproteins and measured their expression and secretion levels in thesupernatant of cells of the human embryonic kidney cell line 293, whichare transformed by adenovirus 5 (Graham et al., J. Gen. Virol. 36:59(1977)). Several high expressers and high level secretory proteins wereobserved.

In one embodiment, secretory leader sequences belonging to the secretedprotein collagen type IX alpha I chain, long form was selected tofurther examine its ability to promote expression and secretion whenused as a heterologous secretory leader sequence. As described herein,the amino acid sequence of the secreted protein collagen type IX alpha Ichain, long form is predicted to be MKTCWKIPVFFFVCSFLEPWASA (SEQ IDNO.:14). As further described herein, vectors were constructedcontaining this particular secretory leader, several proteins werecloned removing the secretory leader from the full length encodingsequence, and by cloning them into vectors containing SEQ ID NO.:14,resulting in secreted proteins with a heterologous secretory leadersequence. High expression and secretion of several other selectedproteins were also observed.

Identified secretory leader sequences, described herein include, forexample, interleukin-9 precursor, T cell growth factor P40, P40cytokine, triacylglycerol lipase, pancreatic precursor, somatoliberinprecursor, vasopressin-neurophysin 2-copeptin precursor,beta-enoendorphin-dynorphin precursor, complement C2 precursor, smallinducible cytokine A14 precursor, elastase 2A precursor, plasma serineprotease inhibitor precursor, granulocyte-macrophage colony-stimulatingfactor precursor, interleukin-2 precursor, interleukin-3 precursor,alpha-fetoprotein precursor, alpha-2-HS-glycoprotein precursor, serumalbumin precursor, inter-alpha-trypsin inhibitor light chain, serumamyloid P-component precursor, apolipoprotein A-II precursor,apolipoprotein D precursor, colipase precursor, carboxypeptidase A1precursor, alpha-s1 casein precursor, beta casein precursor, cystatin SAprecursor, follitropin beta chain precursor, glucagon precursor,complement factor H precursor, histidine-rich glycoprotein precursor,interleukin-5 precursor, alpha-lactalbumin precursor, Von Ebner's glandprotein precursor, matrix Gla-protein precursor, alpha-1-acidglycoprotein 2 precursor, phospholipase A2 precursor, dendritic cellchemokine 1, statherin precursor, transthyretin precursor,apolipoprotein A-1 precursor, apolipoprotein C-III precursor,apolipoprotein E precursor, complement component C8 gamma chainprecursor, serotransferrin precursor, beta-2-microglobulin precursor,neutrophils defensins 1 precursor, triacylglycerol lipase gastricprecursor, haptoglobin precursor, neutrophils defensins 3 precursor,neuroblastoma suppressor of tumorigenicity 1 precursor, small induciblecytokine A13 precursor, CD5 antigen-like precursor, phospholipidstransfer protein precursor, dickkopf related protein-4 precursor,elastase 2B precursor, alpha-1-acid glycoprotein 1 precursor,beta-2-glycoprotein 1 precursor, neutrophil gelatinase-associatedlipocalin precursor, C-reactive protein precursor, interferon gammaprecursor, kappa casein precursor, plasma retinol-binding proteinprecursor, interleukin-13 precursor, and any of the secreted proteinsset forth in the Tables or Sequence Listing.

The secretory leader sequences, vectors, and methods described herein,are useful in the expression of a wide variety of polypeptides,including, for example, secreted polypeptides, extracellular proteins,transmembrane proteins, and receptors, such as soluble receptors.Examples of such polypeptides include, but are not limited to cytokinesand growth factors, such as interleukins 1-18, interferons, lymphokines,hormones, Regulated on Activation, Normal T Expressed and Secreted(RANTES), lymphotoxin-13, Fas ligand, flt-3 ligand, ligand for receptoractivator of NF-kappa B (RANKL), soluble receptors, TNF-relatedapoptosis-inducing ligand (TRAIL), CD40 ligand, 0x40 ligand, 4-1BBligand (and other members of the TNF family), thymic stroma-derivedlymphopoietin, stimulatory factors, for example, granulocyte colonystimulating factor (G-CSF) and granulocyte-macrophage colony stimulatingfactor (GM-CSF), inhibitory factors, mast cell growth factor, stem cellgrowth factor, epidermal growth factor, growth hormone, tumor necrosisfactor (TNF), leukemia inhibitory factor (LIF), oncostatin-M,hematopoietic factors such as erythropoietin and thrombopoietin, andsplice variants of any of these.

Descriptions of some proteins that can be expressed according to theinvention may be found in, for example, Human Cytokines: Handbook forBasic and Clinical Research, Vol. II (Aggarwal and Gutterman, eds.Blackwell Sciences, Cambridge Mass., 1998); Growth Factors: A PracticalApproach (McKay and Leigh, eds., Oxford University Press Inc., New York,1993) and The Cytokine Handbook (A. W. Thompson, ed.; Academic Press,San Diego Calif.; 1991).

Receptors for any of the aforementioned proteins may also be expressedusing secretory leader sequences, vectors and methods described herein,including, for example, both forms of tumor necrosis factor receptor(referred to as p55 and p75), interleukin-1 receptors (type 1 and 2),interleukin-4 receptor, interleukin-15 receptor, interleukin-17receptor, interleukin-18 receptor, granulocyte-macrophage colonystimulating factor receptor, granulocyte colony stimulating factorreceptor, receptors for oncostatin-M and leukemia inhibitory factor,receptor activator of NF-kappa B (RANK), receptors for TRAIL, andreceptors that comprise death domains, such as Fas or apoptosis-inducingreceptor (AIR).

Other proteins that can be expressed using the secretory leadersequences, vectors and methods described herein include, for example,cluster of differentiation antigens (referred to as CD proteins), forexample, those disclosed in Leukocyte Typing VI (Proceedings of the VIthInternational Workshop and Conference; Kishimoto, Kikutani et al., eds.;Kobe, Japan, 1996), or CD molecules disclosed in subsequent workshops.Examples of such molecules include CD27, CD30, CD39, CD40; and ligandsthereto (CD27 ligand, CD30 ligand and CD40 ligand). Several of these aremembers of the TNF receptor family, which also includes 41BB and OX40;the ligands are often members of the TNF family (as are 4-1BB ligand andOX40 ligand); accordingly, members of the TNF and TNFR families can alsobe expressed using the present invention.

Proteins that are enzymatically active may also be expressed employingthe herein described secretory leader sequences, vectors and methods andinclude, for example, metalloproteinase-disintegrin family members,various kinases (including streptokinase and tissue plasminogenactivator as well as death associated kinase containing ankyrin repeats,and IKR 1 and 2), TNF-alpha converting enzyme, and numerous otherenzymes. Ligands for enzymatically active proteins can also be expressedby applying the instant invention.

The secretory leader sequences, vectors, and methods described hereinare also useful for the expression of other types of recombinantproteins, including, for example, immunoglobulin molecules or portionsthereof, and chimeric antibodies (antibodies having a human constantregion couples to a murine antigen binding region) or fragments thereof.Numerous techniques are known by which DNA encoding immunoglobulinmolecules can be manipulated to yield DNAs capable of encodingrecombinant proteins such as single chain antibodies, antibodies withenhanced affinity, or other antibody-based polypeptides (see, forexample, Larrick et al., Biotechnology 7:934-938, 1989; Reichmann etal., Nature 332:323-327, 1988; Roberts et al., Nature 328:731-734, 1987;Verhoeyen et al., Science 239:1534-1536, 1988; and Chaudhary et al.,Nature 339:394-397, 1989).

Co-Translational and Post-Translational Modifications

The invention encompasses polypeptides which are differentially modifiedduring or after translation, for example by glycosylation, acetylation,phosphorylation, amidation, derivatization by known protecting/blockinggroups, proteolytic cleavage, or linkage to an antibody molecule orother cellular ligand. Any of numerous chemical modifications may becarried out by known techniques, including, but not limited to, specificchemical cleavage by cyanogen bromide, trypsin, chymotrypsin, papain, V8protease; NABH₄; acetylation; formylation; oxidation; reduction; and/ormetabolic synthesis in the presence of tunicamycin.

Additional post-translational modifications encompassed by the inventioninclude, for example, for example, N-linked or O-linked carbohydratechains, processing of N-terminal or C-terminal ends), attachment ofchemical moieties to the amino acid backbone, chemical modifications ofN-linked or O-linked carbohydrate chains, and addition or deletion of anN-terminal methionine residue as a result of procaryotic host cellexpression. The polypeptides may also be modified with a detectablelabel, such as an enzymatic, fluorescent, isotopic, or affinity label toallow for detection and isolation of the protein.

Also provided by the invention are chemically modified derivatives ofthe polypeptides of the invention which may provide additionaladvantages such as increased solubility, stability, and circulating timeof the polypeptide, or decreased immunogenicity (see U.S. Pat. No.4,179,337). The chemical moieties for derivitization may be chosen fromwater soluble polymers such as polyethylene glycol, ethyleneglycol/propylene glycol copolymers, carboxymethylcellulose, dextran,polyvinyl alcohol and the like. The polypeptides may be modified atrandom positions within the molecule, or at predetermined positionswithin the molecule and may include one, two, three, or more attachedchemical moieties.

A polymer may be of any molecular weight, and may be branched orunbranched. For polyethylene glycol, a suitable molecular weight isbetween about 1 kDa and about 100 kDa (the term “about” indicating thatin preparations of polyethylene glycol, some molecules will weigh more,some less, than the stated molecular weight) for ease in handling andmanufacturing. Other sizes may be used, depending on the desiredtherapeutic profile (e.g., the duration of sustained release desired,the effects, if any on biological activity, the ease in handling, thedegree or lack of antigenicity and other known effects of thepolyethylene glycol to a therapeutic protein or analog).

The polyethylene glycol molecules (or other chemical moieties) should beattached to the protein with consideration of effects on functional orantigenic domains of the protein. There are a number of attachmentmethods available to those skilled in the art, such as EP 0 401 384(coupling PEG to G-CSF); see also Malik et al., Exp. Hematol.20:1028-1035 (1992) (reporting pegylation of GM-CSF using tresylchloride). For example, polyethylene glycol may be covalently boundthrough amino acid residues via a reactive group, such as a free aminoor carboxyl group. Reactive groups are those to which an activatedpolyethylene glycol molecule may be bound. The amino acid residueshaving a free amino group may include lysine residues and the N-terminalamino acid residues; those having a free carboxyl group may includeaspartic acid residues glutamic acid residues and the C-terminal aminoacid residue. Sulfhydryl groups may also be used as a reactive group forattaching the polyethylene glycol molecules. Suitable for therapeuticpurposes is attachment at an amino group, such as attachment at theN-terminus or lysine group.

One may specifically desire proteins chemically modified at theN-terminus. Using polyethylene glycol as an illustration of the presentcomposition, one may select from a variety of polyethylene glycolmolecules (by molecular weight, branching, etc.), the proportion ofpolyethylene glycol molecules to protein (polypeptide) molecules in thereaction mix, the type of pegylation reaction to be performed, and themethod of obtaining the selected N-terminally pegylated protein. Themethod of obtaining the N-terminally pegylated preparation (i.e.,separating this moiety from other monopegylated moieties if necessary)may be by purification of the N-terminally pegylated material from apopulation of pegylated protein molecules. Selective proteins chemicallymodified at the N-terminus modification may be accomplished by reductivealkylation which exploits differential reactivity of different types ofprimary amino groups (lysine versus the N-terminal) available forderivatization in a particular protein. Under the appropriate reactionconditions, substantially selective derivatization of the protein at theN-terminus with a carbonyl group containing polymer is achieved.

Chromosome Assays

In certain embodiments relating to chromosomal mapping, a cDNA hereindisclosed is used to clone the genomic nucleic acid of MGD-CSF. This canbe accomplished using a variety of well known techniques and libraries,which generally are commercially available. The genomic DNA then is usedfor in situ chromosome mapping using techniques well known for thispurpose. Therefore, the nucleic acid molecules of the present inventionare also valuable for chromosome identification. The sequence isspecifically targeted to and can hybridize with a particular location onan individual human chromosome. Moreover, there is a current need foridentifying particular sites on the chromosome. Few chromosome markingreagents based on actual sequence data (repeat polymorphisms) arepresently available for marking chromosomal location. The mapping ofDNAs to chromosomes according to the present invention is an importantfirst step in correlating those sequences with genes associated withdisease.

Briefly, sequences can be mapped to chromosomes by preparing PCR primersfrom the cDNA. Computer analysis of the 3′ untranslated region is usedto rapidly select primers that do not span more than one exon in thegenomic DNA, thus complicating the amplification process. These primersare then used for PCR screening of somatic cell hybrids containingindividual human chromosomes. Only those hybrids containing the humangene corresponding to the primer will yield an amplified fragment.

PCR mapping of somatic cell hybrids is a rapid procedure for assigning aparticular DNA to a particular chromosome. Using the present inventionwith the same oligonucleotide primers, sublocalization can be achievedwith panels of fragments from specific chromosomes or pools of largegenomic clones in an analogous manner. Other mapping strategies that cansimilarly be used to map to its chromosome include in situhybridization, prescreening with labeled flow-sorted chromosomes andpreselection by hybridization to construct chromosome specific-cDNAlibraries.

Fluorescence in situ hybridization (FISH) of a cDNA clone to a metaphaseChromosomal spread can be used to provide a precise chromosomal locationin one step. This technique can be used with a cDNA as short asapproximately 50-60 bases. For a review of this technique, see Verma etal., Human Chromosomes: A Manual of Basic Techniques, Pergamon Press,New York (1988).

Once a sequence has been mapped to a precise chromosomal location, thephysical position of the sequence on the chromosome can be correlatedwith genetic map data. (Such data are found, for example, in V.McKusick, Mendelian Inheritance in Man, available on line through JohnsHopkins University Welch Medical Library). The relationship betweengenes and diseases that have been mapped to the same chromosomal regionare then identified through linkage analysis (coinheritance ofphysically adjacent genes).

Next, differences can be determined in the cDNA or genomic sequences ofaffected and unaffected individuals. If a mutation is observed in someor all of the affected individuals but not in any normal individuals,then the mutation is likely to be the causative agent of the disease.With current resolution of physical mapping and genetic mappingtechniques, a cDNA precisely localized to a chromosomal regionassociated with the disease could be one of between 50 and 500 potentialcausative genes (assuming 1 megabase mapping resolution and one gene per20 kb).

The gene encoding MGD-CSF is located at chromosome 16q22.1. Linkageanalysis studies suggest that a gene on 16q22 is involved in thecausation of familial myelogenous leukemia (Horwitz et al., Am. J. Hum.Genetics 61:873-881 (1997)). In this study of a family with 11 relevantmeioses transmitting autosomal dominant acute myeloid leukemia andmyelodysplasia, linkages to the well-known leukemia translocationbreakpoint regions 21q22.1-q22.2 and 9p22-p21 were excluded. Horwitz etal., linked these diseases, using the microsatellite marker D16S522,with a maximum 2-point lod score of 2.82 at recombination fractiontheta=0.0, thus providing evidence for linkage to 16q22. Haplotypeanalysis showed a 23.5-cM region of 16q22 that was inherited in commonby all affected family members and extended from D16S451 to D16S289.Nonparametric linkage analysis gave a P-value of 0.00098 for theconditional probability of linkage. Mutational analysis excludedexpansion of the AT-rich minisatellite repeat FRA16B fragile site andthe CAG trinucleotide repeat in the E2F-4 transcription factor, which ispresent in many growth-responsive and growth-promoting genes. The‘repeat expansion detection’ method, capable of detecting dynamicmutation associated with anticipation, more generally excluded large CAGrepeat expansion as a cause of leukemia in this family. MGD-CSF islocated at chromosome 16q22.1. Thus, it may potentially play a role inacute myeloid leukemia and myelodysplasia and may be used to treat thesediseases.

Therapeutic Compositions and Formulations

The polypeptides, agonists, and antagonists of the present invention maybe employed in combination with a suitable pharmaceutical carrier tocomprise a pharmaceutical composition for parenteral administration.Such compositions comprise a therapeutically effective amount of thepolypeptide, agonist, or antagonist and a pharmaceutically acceptablecarrier or excipient. Such a carrier includes, but is not limited to,saline, buffered saline, dextrose, water, glycerol, ethanol, andcombinations thereof. The formulation should suit the mode ofadministration.

The MGD-CSF polypeptide compositions will be formulated and dosed in afashion consistent with good medical practice, taking into account theclinical condition of the individual subject, the site of delivery ofthe MGD-CSF polypeptide composition, the method of administration, thescheduling of administration, and other factors known to practitioners.The effective amount of MGD-CSF polypeptide for purposes herein is thusdetermined by such considerations.

The invention also provides a pharmaceutical pack or kit comprising oneor more containers filled with one or more of the ingredients of thepharmaceutical compositions of the invention. Associated with suchcontainer(s) can be a notice in the form prescribed by a governmentalagency regulating the manufacture, use or sale of pharmaceuticals orbiological products, which notice reflects approval by the agency ofmanufacture, use or sale for human administration. In addition, thepolypeptides, agonists and antagonists of the present invention may beemployed in conjunction with other therapeutic compounds.

The pharmaceutical compositions may be administered in a convenientmanner such as by the oral, topical, intravenous, intraperitoneal,intramuscular, subcutaneous, intranasal, or intradermal routes. Thepharmaceutical compositions are administered in an amount which iseffective for treating and/or prophylaxis of the specific indication. Ingeneral, they are administered in an amount of at least about 10micrograms/kg body weight and in most cases they will be administered inan amount not in excess of about 8 milligrams/kg body weight per day.

The polypeptides of the invention, and agonist and antagonist compoundswhich are polypeptides, may also be employed in accordance with thepresent invention by expression of such polypeptides in vivo, i.e., genetherapy. Thus, for example, cells may be engineered with apolynucleotide (DNA or RNA) encoding for the polypeptide ex vivo; theengineered cells are then provided to a patient. Such methods arewell-known in the art. For example, cells may be engineered byprocedures known in the art by use of a retroviral particle containingRNA encoding for the polypeptide of the present invention.

Similarly, cells may be engineered in vivo for expressing thepolypeptide in vivo, for example, by procedures known in the art. Asknown in the art, a cell producing a retroviral particle containing RNAencoding the polypeptide of the present invention may be administered toa patient for the purpose of engineering cells in vivo and expressingthe polypeptide in vivo. These and other methods for administering apolypeptide of the present invention by similar methods should beapparent to those skilled in the art from the teachings of the presentinvention. For example, the expression vehicle for engineering cells maybe other than a retroviral particle, for example, an adenovirus, whichmay be used to engineer cells in vivo after combination with a suitabledelivery vehicle.

Retroviruses from which the retroviral plasmid vectors hereinabovementioned may be derived include, but are not limited to, Moloney murineleukemia virus, spleen necrosis virus, Rous sarcoma virus, Harveysarcoma virus, avian leukosis virus, gibbon ape leukemia virus, humanimmunodeficiency virus, adenovirus (HIV), myeloproliferative sarcomavirus, and mammary tumor virus.

The nucleic acid sequence encoding the polypeptide of the presentinvention is under the control of a suitable promoter. Vectors of theinvention include one or more promoters. Suitable promoters which may beemployed include, but are not limited to, the retroviral long terminalrepeat (LTR); the SV40 promoter; and the human cytomegalovirus (CMV)promoter described in Miller, et al., Biotechniques, Vol. 7, No. 9,980-990 (1989), or any other homologous or heterologous promoter, forexample, cellular promoters such as eukaryotic cellular promotersincluding, but not limited to, the histone, pol III, and β-actinpromoters. Other viral promoters which may be employed include, but arenot limited to, adenovirus promoters, for example, the adenoviral majorlate promoter; thymidine kinase (TK) promoters; and B19 parvoviruspromoters.

Suitable promoters include, but are not limited to, the respiratorysyncytial virus (RSV) promoter; inducible promoters, such as the MMTpromoter, the metallothionein promoter; heat shock promoters; thealbumin promoter; the ApoA1 promoter; human globin promoters; viralthymidine kinase promoters, such as the herpes simplex thymidine kinasepromoter; retroviral LTRs (including the modified retroviral LTRshereinabove described); the beta-actin promoter; and human growthhormone promoters. The promoter also may be the native promoter whichcontrols the gene encoding the polypeptide. The selection of a suitablepromoter will be apparent to those skilled in the art from the teachingscontained herein.

A retroviral plasmid vector can be employed to transduce packaging celllines to form producer cell lines. Examples of packaging cells which maybe transfected include, but are not limited to, the PE501, PA317, PA12,T19-14X, VT-19-17-H2, CRE, CRIP, GP+E-86, GP+envAm12, and DAN cell linesas described in Miller, Human Gene Therapy, 1:5-14 (1990). The vectormay transduce the packaging cells through any means known in the art.Such means include, but are not limited to, electroporation, the use ofliposomes, and CaPO₄ precipitation. In one alternative, the retroviralplasmid vector may be encapsulated into a liposome, or coupled to alipid, and then administered to a host.

The producer cell line generates infectious retroviral vector particleswhich include the nucleic acid sequence(s) encoding the polypeptides.Such retroviral vector particles then may be employed to transduceeukaryotic cells, either in vitro or in vivo. The transduced eukaryoticcells will express the nucleic acid sequence(s) encoding thepolypeptide. Eukaryotic cells which may be transduced include, but arenot limited to, embryonic stem cells, embryonic carcinoma cells, as wellas hematopoietic stem cells, hepatocytes, fibroblasts, myoblasts,keratinocytes, endothelial cells, and bronchial epithelial cells.

In some embodiments, MGD-CSF compositions are provided in formulationwith pharmaceutically acceptable excipients, a wide variety of which areknown in the art (Gennaro, Remington: The Science and Practice ofPharmacy with Facts and Comparisons: Drugfacts Plus, 20th ed. (2003);Ansel et al., Pharmaceutical Dosage Forms and Drug Delivery Systems,7^(th) ed., Lippencott Williams and Wilkins (2004); Kibbe et al.,Handbook of Pharmaceutical Excipients, 3^(rd) ed., Pharmaceutical Press(2000)). Pharmaceutically acceptable excipients, such as vehicles,adjuvants, carriers or diluents, are available to the public. Moreover,pharmaceutically acceptable auxiliary substances, such as pH adjustingand buffering agents, tonicity adjusting agents, stabilizers, wettingagents and the like, are available to the public.

In pharmaceutical dosage forms, the compositions of the invention can beadministered in the form of their pharmaceutically acceptable salts, orthey can also be used alone or in appropriate association, as well as incombination, with other pharmaceutically active compounds. The subjectcompositions are formulated in accordance to the mode of potentialadministration. Administration of the agents can be achieved in variousways, including oral, buccal, nasal, rectal, parenteral,intraperitoneal, intradermal, transdermal, subcutaneous, intravenous,intra-arterial, intracardiac, intraventricular, intracranial,intratracheal, and intrathecal administration, etc., or otherwise byimplantation or inhalation. Thus, the subject compositions can beformulated into preparations in solid, semi-solid, liquid or gaseousforms, such as tablets, capsules, powders, granules, ointments,solutions, suppositories, enemas, injections, inhalants and aerosols.The following methods and excipients are merely exemplary and are in noway limiting.

Compositions for oral administration can form solutions, suspensions,tablets, pills, granules, capsules, sustained release formulations, oralrinses, or powders. For oral preparations, the agents, polynucleotides,and polypeptides can be used alone or in combination with appropriateadditives, for example, with conventional additives, such as lactose,mannitol, corn starch, or potato starch; with binders, such ascrystalline cellulose, cellulose derivatives, acacia, corn starch, orgelatins; with disintegrators, such as corn starch, potato starch, orsodium carboxymethylcellulose; with lubricants, such as talc ormagnesium stearate; and if desired, with diluents, buffering agents,moistening agents, preservatives, and flavoring agents.

Suitable excipient vehicles are, for example, water, saline, dextrose,glycerol, ethanol, or the like, and combinations thereof. In addition,if desired, the vehicle can contain minor amounts of auxiliarysubstances such as wetting or emulsifying agents or pH buffering agents.Actual methods of preparing such dosage forms are known, or will beapparent, to those skilled in the art (Gennaro, supra). The compositionor formulation to be administered will contain a quantity of the agentadequate to achieve the desired state in the subject being treated.

The agents, polynucleotides, and polypeptides can be formulated intopreparations for injection by dissolving, suspending, or emulsifyingthem in an aqueous or nonaqueous solvent, such as vegetable or othersimilar oils, synthetic aliphatic acid glycerides, esters of higheraliphatic acids or propylene glycol; and if desired, with conventionaladditives such as solubilizers, isotonic agents, suspending agents,emulsifying agents, stabilizers and preservatives. Other formulationsfor oral or parenteral delivery can also be used, as conventional in theart.

The antibodies, agents, polynucleotides, and polypeptides can beutilized in aerosol formulation to be administered via inhalation. Thecompounds of the present invention can be formulated into pressurizedacceptable propellants such as dichlorodifluoromethane, propane,nitrogen, and the like. Further, the agent, polynucleotides, orpolypeptide composition may be converted to powder form foradministration intranasally or by inhalation, as conventional in theart.

Furthermore, the agents can be made into suppositories by mixing with avariety of bases such as emulsifying bases or water-soluble bases. Thecompounds of the present invention can be administered rectally via asuppository. The suppository can include vehicles such as cocoa butter,carbowaxes and polyethylene glycols, which melt at body temperature, yetare solidified at room temperature.

A polynucleotide, polypeptide, or other modulator, can also beintroduced into tissues or host cells by other routes, such as viralinfection, microinjection, or vesicle fusion. For example, expressionvectors can be used to introduce nucleic acid compositions into a cellas described above. Further, jet injection can be used for intramuscularadministration (Furth et al., Anal. Biochem. 205:365-368 (1992)). TheDNA can be coated onto gold microparticles, and delivered intradermallyby a particle bombardment device, or “gene gun” as described in theliterature (Tang et al., Nature 356:152-154 (1992)), where goldmicroprojectiles are coated with the DNA, then bombarded into skincells.

Unit dosage forms for oral or rectal administration such as syrups,elixirs, and suspensions can be provided wherein each dosage unit, forexample, teaspoonful, tablespoonful, tablet, or suppository, contains apredetermined amount of the composition containing one or more agents.Similarly, unit dosage forms for injection or intravenous administrationcan comprise the agent(s) in a composition as a solution in sterilewater, normal saline, or another pharmaceutically acceptable carrier.

Identification of Agonists and Antagonists

The invention provides modulators, including polypeptides,polynucleotides, and other agents that increase or decrease the activityof their target. Modulators of the invention may act as an agonist orantagonist, and may interfere with the binding or activity ofpolypeptides or polynucleotides. Such modulators, or agents, include,for example, polypeptide variants, whether agonist or antagonist;antibodies, whether agonist or antagonist; soluble receptors, usuallyantagonists; small molecule drugs, whether agonist or antagonist; RNAi,usually an antagonist; antisense molecules, usually an antagonist; andribozymes, usually an antagonist. In some embodiments, an agent is asubject polypeptide, where the subject polypeptide itself isadministered to an individual. In some embodiments, an agent is anantibody specific for a subject “target” polypeptide. In someembodiments, an agent is a chemical compound, such as a small molecule,that may be useful as an orally available drug. Such modulation includesthe recruitment of other molecules that directly effect the modulation.For example, an antibody that modulates the activity of a subjectpolypeptide that is a receptor on a cell surface may bind to thereceptor and fix complement, activating the complement cascade andresulting in lysis of the cell. An agent which modulates a biologicalactivity of a subject polypeptide or polynucleotide increases ordecreases the activity or binding at least about 10%, at least about15%, at least about 20%, at least about 25%, at least about 50%, atleast about 80%, or at least about 2-fold, at least about 5-fold, or atleast about 10-fold or more when compared to a suitable control.

The invention also provides a method of screening compounds to identifythose which modulate the biological activity of a polypeptide of thepresent invention. Examples of the biological activities of thepolypeptides of the invention are described in greater detail herein,for example in the Examples and the Figures.

The invention further provides a method wherein a mammalian cell ormembrane preparation expressing a receptor for a polypeptide of thepresent invention, as described above, is incubated with a labeledpolypeptide of the present invention in the presence of the compound.The ability of the compound to enhance or block this interaction is thenmeasured. Alternatively, the response of a known second messenger systemfollowing interaction of a compound to be screened and a MGD-CSFreceptor is measured and the ability of the compound to bind to thereceptor and elicit a second messenger response is measured to determineif the compound is a potential agonist or antagonist. Such secondmessenger systems include, but are not limited to, those mediated bycAMP, guanylate cyclase, ion channels, and phosphoinositide hydrolysis.

Examples of antagonistic compounds include antibodies, or in some cases,oligonucleotides, which bind to a receptor of a polypeptide of thepresent invention but elicit no second messenger response, or which bindto the MGD-CSF polypeptide itself. Alternatively, a potential antagonistmay be a mutant form of the polypeptide which binds to the receptors butelicits no second messenger response, thus effectively blocking theaction of the polypeptide.

Another compound antagonistic to MGD-CSF genes and gene products is anantisense construct prepared using antisense technology. Antisensetechnology can be used to control gene expression through triple-helixformation or antisense DNA or RNA; both methods are based on the bindingof a polynucleotide to DNA or RNA. For example, a 5′ coding portion ofthe polynucleotide sequence, which encodes mature polypeptides of thepresent invention, can be used to design an antisense RNAoligonucleotide of from about 10 to about 40 base pairs in length. A DNAoligonucleotide is designed to be complementary to a region of the geneinvolved in transcription, for example, a triple helix; see Lee et al.,Nucl. Acids Res., 6:3073 (1979); Cooney et al., Science, 241:456 (1988);and Dervan et al., Science, 251:1360 (1991); thereby preventingtranscription and the production of the polypeptides of the presentinvention. The antisense RNA oligonucleotide hybridizes to the mRNA invivo and blocks translation of the mRNA molecule into the polypeptide,as described by Okano, J. Neurochem., 56:560 (1991);Oligodeoxynucleotides as Antisense Inhibitors of Gene Expression, CRCPress, Boca Raton, Fla. (1988). The oligonucleotides described above canalso be delivered to cells such that the antisense RNA or DNA isexpressed in vivo to inhibit polypeptide production.

Potential antagonist compounds also include small molecules which bindto and occupy the binding site of the receptors, thereby making thereceptor inaccessible to its polypeptide such that normal biologicalactivity is prevented. Examples of small molecules include, but are notlimited to, small peptides or peptide-like molecules. Antagonistcompounds may be employed to inhibit the effects of the polypeptides ofthe invention, described in further detail in the Examples and Figures.The antagonists may be employed to diagnose, determine a prognosis for,prevent, and treat immune-related diseases, as described in furtherdetail below.

The present invention also provides methods for identifying agents, suchas antibodies, which enhance or block the actions of MGD-CSF moleculeson cells. For example, these agents may enhance or block interaction ofMGD-CSF-binding molecules, such as receptors. Agents of interest includeboth agonists and antagonists. The invention provides agonists whichincrease the natural biological functions of MGD-CSF or which functionin a manner similar to MGD-CSF. The invention also provides antagonists,which decrease or eliminate the functions of MGD-CSF.

One method of identifying MGD-CSF agonists and antagonists involvesbiochemical assays following subcellular fractionation. For example, acellular compartment, such as a membrane or cytosolic preparation may beprepared from a cell that expresses a molecule that binds MGD-CSFmolecules, such as a molecule of a signaling or regulatory pathwaymodulated by MGD-CSF molecules. Subcellular fractionation methods areknown in the art of cell biology, and can be tailored to produce crudefractions with discrete and defined components, for example, organellesor organellar membranes. The preparation is incubated with labeledMGD-CSF molecules in the absence or the presence of a candidate moleculewhich may be an MGD-CSF agonist or antagonist. The ability of thecandidate molecule to interact with the binding molecule or an MGD-CSFmolecules is reflected in decreased binding of the labeled ligand.Molecules which bind gratuitously, that is, without inducing the effectsof MGD-CSF molecules, are most likely antagonists. Molecules that bindwell and elicit effects that are the same as or closely related toMGD-CSF molecules may potentially prove to be agonists.

The effects of potential agonists and antagonists may by measured, forinstance, by determining an activity of one or more components of asecond messenger system following interaction of the candidate moleculewith a cell or appropriate cell preparation, and comparing the effectwith that of MGD-CSF molecules, or with that of molecules that elicitthe same effects as MGD-CSF. Second messenger systems which may beuseful in this regard include, but are not limited to, cAMP, cGMP, ionchannels, and phosphoinositide hydrolysis second messenger systems.

Another example of an assay for the identification of MGD-CSFantagonists is a competitive assay that combines a mixture of MGD-CSFmolecules and a potential antagonist, with membrane-bound MGD-CSFreceptor molecules. Under appropriate conditions for a competitiveinhibition assay, this assay can also be performed with recombinantMGD-CSF receptor molecules. MGD-CSF molecules can be labeled, such as byradioactivity, such that the number of MGD-CSF molecules bound to areceptor molecule can be determined accurately to assess theeffectiveness of the potential antagonist.

Potential antagonists include small organic molecules, polypeptides, andantibodies that bind to a polypeptide of the invention, and therebyinhibit or extinguish its activity. Potential antagonists also may besmall organic molecules, polypeptides such as closely related proteinsor antibodies that bind the same sites on a binding molecule, such as areceptor molecule, without inducing MGD-CSF-induced activities, therebypreventing the action of MGD-CSF molecules by excluding MGD-CSFmolecules from binding. Antagonists of the invention include fragmentsof the MGD-CSF molecules having the nucleic acid and amino acidsequences shown in the Sequence Listing.

Other potential antagonists include antisense molecules. Antisensetechnology can be used to control gene expression through, for example,antisense DNA or RNA, or through triple-helix formation. Antisensetechniques are discussed, for example, in Okano, J. Neurochem., 56:560(1991); Oligodeoxynucleotides as Antisense Inhibitors of GeneExpression, CRC Press, Boca Raton, Fla. (1988). Triple helix formationis discussed in, for instance, Lee et al., Nucleic Acids Research,6:3073 (1979); Cooney et al., Science, 241:456 (1988); and Dervan etal., Science, 251:1360 (1991). The methods are based on the binding of apolynucleotide to a complementary DNA or RNA. For example, the 5′ codingportion of a polynucleotide that encodes the mature polypeptide of thepresent invention may be used to design an antisense RNA oligonucleotideof from about 10 to about 40 base pairs in length. A DNA oligonucleotideis designed to be complementary to a region of the gene involved intranscription, thereby preventing transcription and the subsequentproduction of MGD-CSF molecules. The antisense RNA oligonucleotidehybridizes to the mRNA in vivo and blocks translation of the mRNAmolecule into a MGD-CSF polypeptide. The oligonucleotides describedabove can also be delivered to cells such that the antisense RNA or DNAmay be expressed in vivo to inhibit production of MGD-CSF molecules.

Diagnosis

This invention is also related to the use of the genes and gene productsof the present invention as part of a diagnostic assay for detectingdiseases or susceptibility to diseases related to the presence ofmutations in the nucleic acid sequences encoding the polypeptide of thepresent invention. Individuals carrying mutations in a gene of thepresent invention may be detected at the DNA level by a variety oftechniques. Nucleic acids for diagnosis may be obtained from a patient'scells, such as, for example, from blood, urine, saliva, tissue biopsy,and autopsy material. The genomic DNA may be used directly for detectionor may be amplified enzymatically by using PCR, for example, asdescribed by Saiki et al., Nature, 324: 163-166 (1986), prior toanalysis. RNA or cDNA may also be used for the same purpose. As anexample, PCR primers complementary to the nucleic acid encoding apolypeptide of the present invention can be used to identify and analyzemutations. For example, deletions and insertions can be detected by achange in size of the amplified product in comparison to the normalgenotype. Point mutations can be identified by hybridizing amplified DNAto radiolabeled RNA or alternatively, radiolabeled antisense DNAsequences. Perfectly matched sequences can be distinguished frommismatched duplexes by RNase A digestion or by differences in meltingtemperatures.

Genetic testing based on DNA sequence differences may be achieved bydetecting alterations in electrophoretic mobility of DNA fragments ingels run with or without denaturing agents. Small sequence deletions andinsertions can be visualized by high resolution gel electrophoresis. DNAfragments of different sequences may be distinguished on denaturingformamide gradient gels in which the mobilities of different DNAfragments are retarded in the gel at different positions according totheir specific melting or partial melting temperatures, for example, asdescribed by Myers et al., Science, 230:1242 (1985).

Sequence changes at specific locations may also be revealed by nucleaseprotection assays, such as RNase and S1 protection or the chemicalcleavage method as shown in Cotton et al., Proc. Natl. Acad. Sci.,85:4397-4401 (1985). Thus, the detection of a specific DNA sequence maybe achieved by methods such as hybridization, RNase protection, chemicalcleavage, direct DNA sequencing or the use of restriction enzymes, forexample, Restriction Fragment Length Polymorphisms (RFLP) and Southernblotting of genomic DNA. In addition to more conventionalgel-electrophoresis and DNA sequencing, mutations can also be detectedby in situ analysis.

The present invention also relates to a diagnostic assay for detectingaltered levels of MGD-CSF proteins in various tissues. Anover-expression of these proteins compared to normal control tissuesamples may detect the presence of abnormal cellular proliferation, forexample, a tumor. Assays used to detect protein levels in a host-derivedsample are well-known to those of skill in the art and includeradioimmunoassays, competitive-binding assays, Western Blot analysis,ELISA assays, “sandwich” assays, and other assays for the expressionlevels of the genes encoding the MGD-CSF proteins known in the art.Expression can be assayed by qualitatively or quantitatively measuringor estimating the level of MGD-CSF protein, or the level of mRNAencoding MGD-CSF protein, in a biological sample. Assays may beperformed directly, for example, by determining or estimating absoluteprotein level or mRNA level, or relatively, by comparing the MGD-CSFprotein or mRNA to a second biological sample. In performing theseassays, the MGD-CSF protein or mRNA level in the first biological sampleis measured or estimated and compared to a standard MGD-CSF proteinlevel or mRNA level; suitable standards include second biologicalsamples obtained from an individual not having the disorder of interest.Standards may be obtained by averaging levels of MGD-CSF in a populationof individuals not having a disorder related to MGD-CSF expression. Aswill be appreciated in the art, once a standard MGD-CSF protein level ormRNA level is known, it can be used repeatedly as a standard forcomparison.

An ELISA assay, for example, as described by Coligan, et al., CurrentProtocols in Immunology, 1(2), Chap. 6, (1991), utilizes an antibodyprepared with specificity to a polypeptide antigen of the presentinvention. In addition, a reporter antibody is prepared against themonoclonal antibody. To the reporter antibody is attached a detectablereagent such as a radioactive tag, a fluorescent tag, or an enzymatictag, e.g., a horseradish peroxidase. A sample is removed from a host andincubated on a solid support, e.g. a polystyrene dish, that binds theproteins in the sample. Any free protein binding sites on the dish arethen covered by incubating with a non-specific protein, e.g., bovineserum albumin. Next, the specific antibody, e.g., a monoclonal antibody,is incubated in the dish, during which time the antibody attaches to anypolypeptides of the present invention attached to the polystyrene dish.All unbound monoclonal antibody is washed out with buffer. The reporterantibody, for example, one linked to horseradish peroxidase is placed inthe dish, resulting in the binding of the reporter antibody to anyantibody bound to the protein of interest; unattached reporter antibodyis then removed. Substrate, e.g., peroxidase, is then added to the dish,and the amount of signal produced color, e.g., developed in a given timeperiod provides a measurement of the amount of a polypeptide of thepresent invention present in a given volume of patient sample whencompared against a standard.

A competition assay may be employed wherein antibodies specific to apolypeptide of the present invention are attached to a solid support,and labeled MGD-CSF, along with a sample derived from the host, arepassed over the solid support. The label can be detected and quantified,for example, by liquid scintillation chromatography, and the measurementcan be correlated to the quantity of the polypeptide of interest presentin the sample. A “sandwich” assay, similar to an ELISA assay, may beemployed, wherein a polypeptide of the present invention is passed overa solid support and binds to antibody modules attached to the solidsupport. A second antibody is then bound to the polypeptide of interest.A third antibody, which is labeled and specific to the second antibodyis then passed over the solid support and binds to the second antibody.The amount of antibody binding can be quantified; it correlates with theamount of the polypeptide of interest. See,e.g., U.S. Pat. No.4,376,110.

Biological samples of the invention can include any biological sampleobtained from a subject, body fluid, cell line, tissue culture, or othersource which contains MGD-CSF protein or mRNA. As indicated, biologicalsamples include body fluids (such as sera, plasma, urine, synovialfluid, and spinal fluid) which contain free MGD-CSF protein, ovarian orrenal system tissue, and other tissue sources found to express completeor mature MGD-CSF polypeptide or an MGD-CSF receptor. Methods forobtaining tissue biopsies and body fluids from mammals are well known inthe art. Where the biological sample is to include mRNA, a tissue biopsymay provide the source.

Total cellular RNA can be isolated from a biological sample using anysuitable technique such as the single-stepguanidinium-thiocyanate-phenol-chloroform method described inChomczynski and Sacchi, Anal. Biochem., 162:156-159 (1987). Levels ofmRNA encoding the MGD-CSF protein are then assayed using any appropriatemethod. These include Northern blot analysis, S1 nuclease mapping, thepolymerase chain reaction (PCR), reverse transcription in combinationwith the polymerase chain reaction (RT-PCR), and reverse transcriptionin combination with the ligase chain reaction (RT-LCR).

Total cellular RNA can be isolated from a biological sample using anysuitable technique such as the single-stepguanidinium-thiocyanate-phenol-chloroform method described inChomczynski and Sacchi, Anal. Biochem., 162:156-159 (1987). Levels ofmRNA encoding the MGD-CSF protein are then assayed using any appropriatemethod. These include Northern blot analysis, S1 nuclease mapping, PCR,reverse transcription in combination with PCR (RT-PCR), and reversetranscription in combination with the ligase chain reaction (RT-LCR).

Assaying MGD-CSF protein levels in a biological sample can be performedusing antibody-based techniques. For example, MGD-CSF protein expressionin tissues can be studied with classical immunohistological methods, forexample, Jalkanen, M., et al., J. Cell. Biol., 101:976-985 (1985);Jalkanen, M., et al., J. Cell. Biol., 105:3087-3096 (1987). Otherantibody-based methods useful for detecting MGD-CSF protein geneexpression include immunoassays, such as the enzyme linked immunosorbentassay (ELISA) and the radioimmunoassay (RIA). Suitable antibody assaylabels are known in the art and include enzyme labels, such as glucoseoxidase, radioisotopes, and fluorescent labels, such as fluorescein andrhodamine, and biotin.

In addition to assaying MGD-CSF protein levels in a biological sampleobtained from an individual, MGD-CSF protein can also be detected invivo by imaging. Antibody labels or markers for in vivo imaging ofMGD-CSF protein include those detectable by X-radiography, NMR, or ESR.For X-radiography, suitable labels include radioisotopes such as bariumor cesium, which emit detectable radiation but are not overtly harmfulto a subject. Suitable markers for NMR and ESR include those with adetectable characteristic spin, such as deuterium, which may beincorporated into the antibody by labeling of nutrients for the relevanthybridoma.

A MGD-CSF protein-specific antibody or antibody fragment which has beenlabeled with an appropriate detectable imaging moiety, such as aradioisotope, a radio-opaque substance, or a material detectable bynuclear magnetic resonance, is introduced, for example, parenterally,subcutaneously or intraperitoneally, into the subject to be examined foran immune system disorder. It will be understood in the art that thesize of the subject and the imaging system used will determine thequantity of imaging moiety needed to produce diagnostic images. Thelabeled antibody or antibody fragment will then accumulate at thelocation of cells which contain MGD-CSF protein. In vivo tumor imagingis described in Burchiel et al., ed., Chapter 13, Tumor Imaging: TheRadiochemical Detection of Cancer, Masson Publishing, Inc. (1982).

Therapeutic Uses of MGD-CSF Molecules, Agonists, and Antagonists

Molecules of the invention and fragments and variants thereof may beused in diagnosing, prognosing, preventing, treating, and developingtreatments for any disorder mediated, either directly or indirectly, bydefective or insufficient amounts of MGD-CSF. MGD-CSF polypeptides,agonists, or antagonists may be administered to a patient afflicted withsuch a disorder. A gene therapy approach may be applied to treat suchdisorders. Disclosure herein of sequences of the invention permits thedetection of defective MGD-CSF related genes, and the replacementthereof with normal or corrective genes. Defective genes may be detectedin in vitro diagnostic assays, and by comparison of the sequences of theinvention with that of a gene derived from a patient suspected ofharboring a defect.

Molecules of the invention, such as recombinant MGD-CSF may havemultiple effects on the proliferation of different cell types and mayhave multiple effects on the proliferation and differentiation of thesame cell type under different conditions. Under conditions whereinMGD-CSF inhibits proliferation and/or differentiation, recombinantMGD-CSF or related molecules may be used to treat diseases characterizedby abnormal proliferation and/or differentiation. Under conditionswherein MGD-CSF promotes proliferation and/or differentiation, agentsinhibitory to MGD-CSF or related molecules may be used to treat diseasescharacterized by abnormal proliferation and/or differentiation. Suitableinhibitors are described herein, and may include inhibitory antibodies,small molecule inhibitors, antisense oligonucleotides, siRNA, andsoluble receptors.

Disease Applications

The molecules of the invention are useful for treating cancer, immunediseases, such as an autoimmune disease or an inflammatory disease,ischemic diseases, infectious diseases, bone diseases, and neuraldiseases. The molecules of the invention are useful for inhibiting themultiplication of a tumor cell or cancer cell, and for treating cancer.The molecules of the invention can be used accordingly in a variety ofsettings for the treatment of animal cancers. Other particular types ofcancers that can be treated with molecules of the invention include, butare not limited to, those disclosed below.

MGD-CSF may play a role in the retention, proliferation, and survival ofhematopoietic cells in the bone marrow. Therefore, it may be useful inthe treatment of hemaptopoietic cell (for example, neutrophil)deficiency in cancer patients receiving chemotherapy or radiotherapy.

The molecules of the invention may be employed to treatlymphoproliferative disease which results in lymphadenopathy. Themolecules of the invention may mediate apoptosis by stimulating clonaldeletion of T cells and may therefore be employed to treat autoimmunedisease to stimulate peripheral tolerance and cytotoxic T cell mediatedapoptosis. The molecules of the invention may also be employed as aresearch tool in elucidating the biology of allergies and of autoimmunedisorders including systemic lupus erythematosus (SLE), Graves' disease,immunoproliferative disease lymphadenopathy (IPL),angioimmunoproliferative lymphadenopathy (AIL), immunoblastivelymphadenopathy (IBL), rheumatoid arthritis, diabetes, and multiplesclerosis, and to treat graft versus host disease.

The molecules of the invention are useful for killing or inhibiting thereplication of a cell that produces an autoimmune disease or aninflammatory disease or for treating an autoimmune disease or aninflammatory disease. They can be used accordingly in a variety ofsettings for the treatment of an autoimmune disease or an inflammatorydisease in an animal.

The molecules of the invention may also be used to treat, prevent,diagnose and/or determine a prognosis for diseases which include, butare not limited to, autoimmune disorders, immunodeficiency disorders,and graft versus host disease, and recurrent pregnancy loss.Additionally, molecules of the invention may be employed as agents toboost immunoresponsiveness among individuals having a temporary immunedeficiency. Conditions resulting in a temporary immune deficiency thatmay be ameliorated or treated by administering the molecules of theinvention include, but are not limited to, recovery from infectiousdiseases, such as viral infections (for example, influenza, infectiousmononucleosis, or measles), conditions associated with malnutrition,recovery from or conditions associated with stress, recovery from bloodtransfusion, and recovery from surgery.

Molecules of the invention may be used to diagnose, determine aprognosis for, treat, or prevent one or more of the following diseases,disorders, or conditions associated therewith: primaryimmuodeficiencies, immune-mediated thrombocytopenia, Kawasaki syndrome,bone marrow transplant (for example, recent bone marrow transplant inadults or children), chronic B cell lymphocytic leukemia, HIV infection(for example, adult or pediatric HIV infection), chronic inflammatorydemyelinating polyneuropathy, and post-transfusion purpura.

Additionally, molecules of the invention may be used to diagnose,determine a prognosis for, treat or prevent one or more of the followingdiseases, disorders, or conditions associated therewith: Guillain-Barresyndrome, anemia (for example, anemia associated with parvovirus B19,patients with stable multiple myeloma who are at high risk for infection(for example, recurrent infection), autoimmune hemolytic anemia (forexample, warm-type autoimmune hemolytic anemia), thrombocytopenia (forexample, neonatal thrombocytopenia), and immune-mediated neutropenia),transplantation (for example, cytomegalovirus (CMV)-negative recipientsof CMV-positive organs), hypogammaglobulinemia (for example,hypogammaglobulinemic neonates with risk factor for infection ormorbidity), epilepsy (for example, intractable epilepsy), systemicvasculitic syndromes, myasthenia gravis (for example, decompensation inmyasthenia gravis), dermatomyositis, and polymyositis.

Further autoimmune disorders and conditions associated with thesedisorders that may be treated, prevented, diagnosed, and/or have theirprognosis determined by molecules of the invention include, but are notlimited to, autoimmune hemolytic anemia, autoimmune neonatalthrombocytopenia, idiopathic thrombocytopenia purpura,autoimmunocytopenia, hemolytic anemia, antiphospholipid syndrome,dermatitis, allergic encephalomyelitis, myocarditis, relapsingpolychondritis, rheumatic heart disease, glomerulonephritis (forexample, IgA nephropathy), multiple sclerosis, neuritis, uveitisophthalmia, polyendocrinopathies, purpura (for example,Henloch-Scoenlein purpura), Reiter's disease, stiff-man syndrome,autoimmune pulmonary inflammation, Guillain-Barre Syndrome, insulindependent diabetes mellitis, and autoimmune inflammatory eye disease.

Additional autoimmune disorders that may be treated, prevented,diagnosed, and/or have their prognosis determined by molecules of theinvention include but are not limited to autoimmune thyroiditis;hypothyroidism, including Hashimoto's thyroiditis and thyroiditischaracterized, for example, by cell-mediated and humoral thyroidcytotoxicity; SLE (which is often characterized, for example, bycirculating and locally generated immune complexes); Goodpasture'ssyndrome (which is often characterized, for example, by anti-basementmembrane antibodies); pemphigus (which is often characterized, forexample, by epidermal acantholytic antibodies); receptor autoimmunitiessuch as, for example, Graves' disease (which is often characterized, forexample, by antibodies to a thyroid stimulating hormone receptor;myasthenia gravis, which is often characterized, for example, byacetylcholine receptor antibodies); insulin resistance (which is oftencharacterized, for example, by insulin receptor antibodies); autoimmunehemolytic anemia (which is often characterized, for example, byphagocytosis of antibody-sensitized red blood cells); and autoimmunethrombocytopenic purpura (which is often characterized, for example, byphagocytosis of antibody-sensitized platelets).

Further autoimmune disorders which may be treated, prevented, diagnosed,and/or have their prognosis determined by molecules of the inventioninclude but are not limited to rheumatoid arthritis (which is oftencharacterized, for example, by immune complexes in joints); sclerodermawith anti-collagen antibodies (which is often characterized, forexample, by nucleolar and other nuclear antibodies); mixed connectivetissue disease, (which is often characterized, for example, byantibodies to extractable nuclear antigens, for example,ribonucleoprotein); polymyositis/dermatomyositis (which is oftencharacterized, for example, by nonhistone anti-nuclear antibodies);pernicious anemia (which is often characterized, for example, byantiparietal cell, antimicrosome, and anti-intrinsic factor antibodies);idiopathic Addison's disease (which is often characterized, for example,by humoral and cell-mediated adrenal cytotoxicity); infertility (whichis often characterized, for example, by antispermatozoal antibodies);glomerulonephritis (which is often characterized, for example, byglomerular basement membrane antibodies or immune complexes); by primaryglomerulonephritis, by IgA nephropathy; bullous pemphigoid (which isoften characterized, for example, by IgG and complement in the basementmembrane); Sjögren's syndrome (which is often characterized, forexample, by multiple tissue antibodies and/or the specific nonhistoneantinuclear antibody (SS-B)); diabetes mellitus (which is oftencharacterized, for example, by cell-mediated and humoral islet cellantibodies); and adrenergic drug resistance, including adrenergic drugresistance with asthma or cystic fibrosis (which is often characterized,for example, by beta-adrenergic receptor antibodies).

Yet further autoimmune disorders which may be treated, prevented, havetheir prognosis determined by, and/or diagnosed with antagoniststhereof, include, but are not limited to the following disorders:chronic active hepatitis (which is often characterized, for example bysmooth muscle antibodies); primary biliary cirrhosis (which is oftencharacterized, for example, by anti-mitchondrial antibodies); otherendocrine gland failure (which is characterized, for example, byspecific tissue antibodies in some cases); vitiligo (which is oftencharacterized, for example, by anti-melanocyte antibodies); vasculitis(which is often characterized, for example, by immunoglobulin andcomplement in vessel walls and/or low serum complement); post-myocardialinfarction conditions (which are often characterized, for example, byanti-myocardial antibodies); cardiotomy syndrome (which is oftencharacterized, for example, by anti-myocardial antibodies); urticaria(which is often characterized, for example, by IgG and IgM antibodies toIgE); atopic dermatitis (which is often characterized, for example, byIgG and IgM antibodies to IgE); asthma (which is often characterized,for example, by IgG and IgM antibodies to IgE); inflammatory myopathies;and other inflammatory, granulomatous, degenerative, and atrophicdisorders.

In an embodiment, the molecules of the invention, for example,anti-MGD-CSF antibodies, are used to treat or prevent SLE and/orassociated diseases, disorders, or conditions. Lupus-associateddiseases, disorders, and conditions which may be treated or preventedwith molecules of the invention include, but are not limited to,hematologic disorders, for example, hemolytic anemia, leukopenia,lymphopenia, and thrombocytopenia; immunologic disorders, for example,anti-DNA antibodies, and anti-Sm antibodies, rashes, photosensitivity,oral ulcers, arthritis, fever, fatigue, weight loss, serositis, forexample, pleuritus (pleurisy); renal disorders, for example, nephritis;neurological disorders, for example, seizures, peripheral neuropathy andCNS related disorders; gastroinstestinal disorders; Raynaud'sphenomenon; and pericarditis.

The molecules of the invention may also be employed to inhibitneoplasia, such as tumor cell growth. The MGD-CSF polypeptides may beresponsible for tumor destruction through apoptosis and cytotoxicity tocertain cells. Diseases associated with increased cell survival, or theinhibition of apoptosis, that may be treated, prevented, diagnosed,and/or have their prognosis determined by the molecules of the inventioninclude, but are not limited to, cancers (such as follicular lymphomas,carcinomas with p53 mutations, and hormone-dependent tumors, including,but not limited to colon cancer, cardiac tumors, pancreatic cancer,melanoma, retinoblastoma, glioblastoma, lung cancer, intestinal cancer,testicular cancer, stomach cancer, neuroblastoma, myxoma, myoma,lymphoma, endothelioma, osteoblastoma, osteoclastoma, osteosarcoma,chondrosarcoma, adenoma, breast cancer, prostate cancer, Kaposi'ssarcoma and ovarian cancer); autoimmune disorders (such as, multiplesclerosis, Sjögren's syndrome, Graves' disease, Hashimoto's thyroiditis,autoimmune diabetes, biliary cirrhosis, Behcet's disease, Crohn'sdisease, polymyositis, systemic lupus erythematosus and immune-relatedglomerulonephritis, autoimmune gastritis, autoimmune thrombocytopenicpurpura, and rheumatoid arthritis) and viral infections (such as herpesviruses, pox viruses and adenoviruses), inflammation, graft vs. hostdisease (acute and/or chronic), acute graft rejection, and chronic graftrejection. In an embodiment, of the invention are used to inhibitgrowth, progression, and/or metastasis of cancers, in particular thoselisted above or in the paragraph that follows.

Additional diseases or conditions associated with increased cellsurvival, that may be treated, prevented, diagnosed, and/or have theirprognosis determined by the of the invention include, but are notlimited to, progression, and/or metastases of malignancies and relateddisorders such as leukemia (including acute leukemias (for example,acute lymphocytic leukemia, acute myelocytic leukemia, includingmyeloblastic, promyelocytic, myelomonocytic, monocytic, anderythroleukemia)) and chronic leukemias (for example, chronic myelocytic(granulocytic) leukemia and chronic lymphocytic leukemia),myelodysplastic syndrome polycythemia vera, lymphomas (for example,Hodgkin's disease and non-Hodgkin's disease), multiple myeloma,Waldenstrom's macroglobulinemia, heavy chain diseases, and solid tumorsincluding, but not limited to, sarcomas and carcinomas such asfibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenicsarcoma, chordoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma,lymphangioendotheliosarcoma, synovioma, mesothelioma, Ewing's tumor,leiomyosarcoma, rhabdomyosarcoma, colon carcinoma, pancreatic cancer,breast cancer, ovarian cancer, prostate cancer, squamous cell carcinoma,basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceousgland carcinoma, papillary carcinoma, papillary adenocarcinomas,cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renalcell carcinoma, hepatoma, bile duct carcinoma, choriocarcinoma,seminoma, embryonal carcinoma, Wilm's tumor, cervical cancer, testiculartumor, lung carcinoma, small cell lung carcinoma, bladder carcinoma,epithelial carcinoma, glioma, astrocytoma, medulloblastoma,craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acousticneuroma, oligodendroglioma, menangioma, melanoma, neuroblastoma, andretinoblastoma.

Diseases associated with increased apoptosis, that may be treated,prevented, diagnosed, and/or have their prognosis determined bymolecules of the invention include, but are not limited to, AIDS (suchas HIV-induced nephropathy and HIV encephalitis), neurodegenerativedisorders (such as Alzheimer's disease, Parkinson's disease, amyotrophiclateral sclerosis, retinitis pigmentosa, cerebellar degeneration andbrain tumor or prior associated disease), autoimmune disorders such asmultiple sclerosis, Sjögren's syndrome, Graves' disease, Hashimoto'sthyroiditis, autoimmune diabetes, biliary cirrhosis, Behcet's disease,Crohn's disease, polymyositis, systemic lupus erythematosus,immune-related glomerulonephritis, autoimmune gastritis,thrombocytopenic purpura, and rheumatoid arthritis, myelodysplasticsyndromes (such as aplastic anemia), graft vs. host disease (acuteand/or chronic), ischemic injury (such as that caused by myocardialinfarction, stroke and reperfusion injury), liver injury or disease (forexample, hepatitis related liver injury, cirrhosis, ischemia/reperfusioninjury, cholestosis (bile duct injury) and liver cancer), toxin-inducedliver disease (such as that caused by alcohol), septic shock, ulcerativecolitis, cachexia, and anorexia.

Another embodiment of the present invention is directed to the use ofMGD-CSF polynucleotides, polypeptides, or antagonists to reduce MGD-CSFor NP_(—)689669 mediated death of T cells in HIV-infected patients. Therole of T cell apoptosis in the development of AIDS has been the subjectof a number of studies (see, for example, Meyaard et al., Science,257:217-219 (1992); Groux et al., J. Exp. Med., 175:331 (1992); andOyaizu et al., in Cell Activation and Apoptosis in HIV Infection,Andrieu and Lu, eds., Plenum Press, New York, pp. 101-114 (1995)).

It is likely that T cell apoptosis occurs through multiple mechanisms.Fas-mediated apoptosis has been implicated in the loss of T cells in HIVindividuals (Katsikis et al., J. Exp. Med. 181:2029-2036 (1995).Activated human T cells are induced to undergo programmed cell death(apoptosis) upon triggering through the CD3/T cell receptor complex, aprocess termed activated-induced cell death (AICD). AICD of CD4 T cellsisolated from HIV-infected asymptomatic individuals has been reported(Groux et al., supra). Thus, AICD may play a role in the depletion ofCD4+ T cells and the progression to AIDS in HIV-infected individuals.Accordingly, the invention provides a method of inhibitingMGD-CSF-mediated T cell death in HIV patients, comprising administeringmolecules of the invention to the patients. In an embodiment, thepatient is asymptomatic when treatment with MGD-CSF polynucleotides,polypeptides, or antagonists commences. If desired, prior to treatment,peripheral blood T cells may be extracted from an HIV patient, andtested for susceptibility to MGD-CSF-mediated cell death by proceduresknown in the art. In one embodiment, a patient's blood or plasma iscontacted with molecules of the invention, for example, anti-MGD-CSF orNP_(—)689669 antibodies, ex vivo. The antibodies or other antagonistsmay be bound to a suitable chromatography matrix by procedures known inthe art. The patient's blood or plasma flows through a chromatographycolumn containing the antagonist bound to the matrix, before beingreturned to the patient. The immobilized antagonist binds MGD-CSF orNP_(—)689699, thus removing it from the patient's blood.

In additional embodiments, a molecule of the invention is administeredin combination with other inhibitors of T cell apoptosis. For example,as discussed above, Fas-mediated apoptosis also has been implicated inloss of T cells in HIV positive individuals (Katsikis et al., J. Exp.Med., 181:2029-2036 (1995)). Thus, a patient susceptible to both Fasligand mediated and MGD-CSF-mediated T cell death may be treated withboth an agent that blocks MGD-CSF or NP_(—)689699 interactions withtheir receptors and an agent that blocks Fas-ligand/Fas interactions.Suitable agents for blocking binding of Fas-ligand to Fas include, butare not limited to, soluble Fas polypeptides; multimeric forms ofsoluble Fas polypeptides (for example, dimers of sFas/Fc); anti-Fasantibodies that bind Fas without transducing the biological signal thatresults in apoptosis; anti-Fas-ligand antibodies that block binding ofFas-ligand to Fas; and muteins of Fas-ligand that bind Fas but do nottransduce the biological signal that results in apoptosis. Monoclonalantibodies may be employed according to this method. Examples ofsuitable agents for blocking Fas-ligand/Fas interactions, includingblocking anti-Fas monoclonal antibodies, are described in WO 95/10540.

In another example, agents which block binding of MGD-CSF orNP_(—)689669 to a receptor are administered with the molecules of theinvention. Such agents include, but are not limited to, soluble MGD-CSFreceptor polypeptides, multimeric forms of soluble receptorpolypeptides, and MGD-CSF receptor antibodies that bind the MGD-CSF orNP_(—)689669 receptor without transducing the biological signal thatresults in apoptosis, antibodies that block binding of MGD-CSF orNP_(—)689669 to one or more receptors, and muteins that bind toreceptors but do not transduce a biological signal that results inapoptosis.

Molecules of the invention may also be employed to regulatehematopoeisis, including erythropoiesis. Hematopoeisis is a multi-stepcell proliferation and differentiation process which begins with a poolof multipotent stem cells. These cells can proliferate and differentiateinto hematopoietic progenitors in reply to different stimuli. Themolecules of the invention may be used to either stimulate or inhibitdevelopment of hematopoietic cells, for example, erythropoieticprecursor cells.

In an embodiment, the molecules of the invention are used to treat orprevent bone diseases. Molecules of the invention promote thedifferentiation of hematopoeitic stem cells into osteoclastic precursorcells. Accordingly, molecules of the invention can be used to treat bonediseases such as those characterized by defects in osteoclastdifferentiation and function, for example, osteoporosis. MGD-CSF andrelated molecules may be used as therapeutics, for example, proteintherapeutics or in gene therapy, to treat these diseases.

In an embodiment, the molecules of the invention are used to treat orprevent neural diseases. Molecules of the invention promote thedifferentiation of hematopoeitic stem cells into microglial precursorcells. Accordingly, molecules of the invention can be used to treatneural diseases such as those characterized by defects in microglialdifferentiation and function, for example, Alzheimer's disease, multiplesclerosis, acute disseminated encephalomyelopathy, progressivemultifocal leukoencephalopathy, stroke, and Parkinson's disease. MGD-CSFand related molecules may be used as therapeutics, for example, proteintherapeutics or in gene therapy, to treat these diseases.

In an embodiment, the molecules of the invention are used to treat orprevent cardiovascular disorders, including peripheral artery disease,such as limb ischemia. Cardiovascular disorders include cardiovascularabnormalities, such as arterio-arterial fistula, arteriovenous fistula,cerebral arteriovenous malformations, congenital heart defects,pulmonary atresia, and scimitar syndrome. Congenital heart defectsinclude aortic coarctation, cor triatriatum, coronaryvessel anomalies,crisscross heart, dextrocardia, patent ductus arteriosus, Ebstein'sanomaly, Eisenmenger complex, hypoplastic left heart syndrome,levocardia, trilogy of Fallot, tetralogy of Fallot, transposition ofgreat vessels, double outlet right ventricle, tricuspid atresia,persistent truncus arteriosus, and heart septal defects, such asaortopulmonary septal defect, endocardial cushion defects, Lutembacher'sSyndrome, and ventricular heart septal defects.

Cardiovascular disorders which can be treated with molecules of theinvention also include heart disease, such as arrhythmias, carcinoidheart disease, high cardiac output, low cardiac output, cardiactamponade, endocarditis (including bacterial), heart aneurysm, cardiacarrest, congestive heart failure, congestive cardiomyopathy, paroxysmaldyspnea, cardiac edema, heart hypertrophy, congestive cardiomyopathy,left ventricular hypertrophy, right ventricular hypertrophy,post-infarction heart rupture, ventricular septal rupture, heart valvediseases, myocardial diseases, myocardial ischemia, pericardialeffusion, pericarditis (including constrictive and tuberculous),pneumopericardium, postpericardiotomy syndrome, pulmonary heart disease,rheumatic heart disease, ventricular dysfunction, hyperemia,cardiovascular pregnancy complications, scimitar syndrome,cardiovascular syphilis, and cardiovascular tuberculosis.

Arrhythmias that can be treated with molecules of the invention includesinus arrhythmia, atrial fibrillation, atrial flutter, bradycardia,extrasystole, Adams-Stokes syndrome, bundle-branch block, sinoatrialblock, long QT syndrome, parasystole, Lown-Ganong-Levine syndrome,Mahaim-type pre-excitation syndrome, Wolff-Parkinson-White syndrome,sick sinus syndrome, tachycardias, and ventricular fibrillation.Tachycardias that can be treated with molecules of the invention includeparoxysmal tachycardia, supraventricular tachycardia, acceleratedidioventricular rhythm, atrioventricular nodal reentry tachycardia,ectopic atrial tachycardia, ectopic junctional tachycardia, sinoatrialnodal reentry tachycardia, sinus tachycardia, Torsades de Pointes, andventricular tachycardia. Heart valve diseases include aortic valveinsufficiency, aortic valve stenosis, heart murmurs, aortic valveprolapse, mitral valve prolapse, tricuspid valve prolapse, mitral valveinsufficiency, mitral valve stenosis, pulmonary atresia, pulmonary valveinsufficiency, pulmonary valve stenosis, tricuspid atresia, tricuspidvalve insufficiency, and tricuspid valve stenosis.

Myocardial disease that can be treated with molecules of the inventiondiseases also include alcoholic cardiomyopathy, congestivecardiomyopathy, hypertrophic cardiomyopathy, aortic subvalvularstenosis, pulmonary subvalvular stenosis, restrictive cardiomyopathy,Chagas cardiomyopathy, endocardial fibroelastosis, endomyocardialfibrosis, Kearns syndrome, myocardial reperfusion injury, andmyocarditis. Myocardial ischemias that can be treated with molecules ofthe invention include coronary diseases, such as angina pectoris,coronary aneurysm, coronary arteriosclerosis, coronary thrombosis,coronary vasospasm, myocardial infarction, and myocardial stunning.

Cardiovascular diseases that can be treated with molecules of theinvention also include vascular diseases such as aneurysms,angiodysplasia, angiomatosis, bacillary angiomatosis, Hippel-Lindaudisease, Klippel-Trenaunay-Weber syndrome, Sturge-Weber syndrome,angioneurotic edema, aortic diseases, Takayasu's arteritis, aortitis,Leriche's syndrome, arterial occlusive diseases, arteritis, enarteritis,polyarteritis nodosa, cerebrovascular disorders, diabetic angiopathies,diabetic retinopathy, embolisms, thrombosis, erythromelalgia,hemorrhoids, hepatic venoocclusive disease, hypertension, hypotension,ischemia, peripheral vascular diseases, phlebitis, pulmonaryvenoocclusive disease, Raynaud's disease, CREST syndrome, retinal veinocclusion, scimitar syndrome, superior vena cava syndrome,telangiectasia, ataxia telangiectasia, hereditary hemorrhagictelangiectasia, varicocele, varicose veins, varicose ulcer, vasculitis,and venous insufficiency. Aneurysms include dissecting aneurysms, falseaneurysms, infected aneurysms, ruptured aneurysms, aortic aneurysms,cerebral aneurysms, coronary aneurysms, heart aneurysms, and iliacaneurysms.

Arterial occlusive diseases that can be treated with molecules of theinvention include arteriosclerosis, intermittent claudication, carotidstenosis, fibromuscular dysplasias, mesenteric vascular occlusion,Moyamoya disease, renal artery obstruction, retinal artery occlusion,and thromboangiitis obliterans.

Cerebrovascular disorders that can be treated with molecules of theinvention include carotid artery diseases, cerebral amyloid angiopathy,cerebral aneurysm, cerebral anoxia, cerebral arteriosclerosis, cerebralarteriovenous malformation, cerebral artery diseases, cerebral embolismand thrombosis, carotid artery thrombosis, sinus thrombosis,Wallenberg's syndrome, cerebral hemorrhage, epidural hematoma, subduralhematoma, subarachnoid hemorrhage, cerebral infarction, cerebralischemia (including transient), subclavian steal syndrome,periventricular leukomalacia, vascular headache, cluster headache,migraine, and vertebrobasilar insufficiency.

Embolisms that can be treated with molecules of the invention includeair embolisms, amniotic fluid embolisms, cholesterol embolisms, blue toesyndrome, fat embolisms, pulmonary embolisms, and thromboembolisms.Thromboses include coronary thrombosis, hepatic vein thrombosis, retinalvein occlusion, carotid artery thrombosis, sinus thrombosis,Wallenberg's syndrome, and thrombophlebitis.

Ischemias that can be treated with molecules of the invention includecerebral ischemia, ischemic colitis, compartment syndromes, anteriorcompartment syndrome, myocardial ischemia, reperfusion injuries, andperipheral limb ischemia. Vasculitis that can be treated with moleculesof the invention includes aortitis, arteritis, Behcet's syndrome,Churg-Strauss syndrome, mucocutaneous lymph node syndrome,thromboangiitis obliterans, hypersensitivity vasculitis,Schoenlein-Henoch purpura, allergic cutaneous vasculitis, and Wegener'sgranulomatosis.

The present further provides for treatment of diseases associated withneovascularization by administration of the molecules of the invention.Malignant and metastatic conditions which can be treated with themolecules of the invention include, but are not limited to thosemalignancies, solid tumors, and cancers described herein and otherwiseknown in the art (for a review of such disorders, see Fishman et al.,Medicine, 4th ed., J.B. Lippincott Co., Philadelphia (1997)).

Additionally, ocular disorders associated with neovascularization whichcan be treated with molecules of the invention include, but are notlimited to, neovascular glaucoma, diabetic retinopathy, retinoblastoma,retrolental fibroplasia, uveitis, retinopathy of prematurity, maculardegeneration, corneal graft neovascularization, as well as other eyeinflammatory diseases, ocular tumors, and diseases associated withchoroidal or iris neovascularization. See, for example, reviews byWaltman et al., Am. J. Ophthal., 85:704-710 (1978) and Gartner et al.,Surv. Ophthal., 22:291-312 (1978).

Additionally, disorders which can be treated with molecules of theinvention include, but are not limited to, hemangioma, arthritis,psoriasis, angiofibroma, atherosclerotic plaques, delayed wound healing,granulations, hemophilic joints, hypertrophic scars, nonunion fractures,Osler-Weber syndrome, pyogenic granuloma, scleroderma, trachoma, andvascular adhesions.

Molecules of the invention antagonists thereof, are useful in thediagnosis and treatment or prevention of a wide range of diseases and/orconditions, including, but not limited to, cancer (for example, immunecell related cancers, breast cancer, prostate cancer, ovarian cancer,follicular lymphoma, cancer associated with mutation or alteration ofp53, brain tumor, bladder cancer, uterocervical cancer, colon cancer,colorectal cancer, non-small cell carcinoma of the lung, and small cellcarcinoma of the lung, stomach cancer, etc.). They are also useful inthe diagnosis and treatment or prevention of lymphoproliferativedisorders (for example, lymphadenopathy), microbial disorders (forexample, viral, bacterial, etc.), infections, for example, HIV-1infection, HIV-2 infection, herpesvirus infection (including, but notlimited to, HSV-1, HSV-2, CMV, VZV, HHV-6, HHV-7, EBV), adenovirusinfection, poxvirus infection, human papilloma virus infection,hepatitis infection (for example, HAV, HBV, HCV, etc.), Helicobacterpylori infection, invasive Staphylococci, etc.), and parasiticinfection. They are further useful in the diagnosis and treatment orprevention of nephritis, bone disease (for example, osteoporosis),atherosclerosis, pain, cardiovascular disorders (for example,neovascularization, hypovascularization), and reduced circulation (forexample, ischemic diseases, such as myocardial infarction, stroke, etc.,AIDS, allergy, inflammation, neurodegenerative disease (for example,Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis,pigmentary retinitis, cerebellar degeneration, etc., graft rejection(acute and chronic), graft vs. host disease, diseases resulting fromosteomyelodysplasia (for example, aplastic anemia, etc.), joint tissuedestruction in rheumatism, liver disease (for example, acute and chronichepatitis, liver injury, biliary disease, and cirrhosis), autoimmunedisease (for example, multiple sclerosis, rheumatoid arthritis, SLE,immune complex glomerulonephritis, autoimmune diabetes, autoimmunethrombocytopenic purpura, Graves' disease, Hashimoto's thyroiditis,etc.), cardiomyopathy (for example, dilated cardiomyopathy), diabetes,diabetic complications (for example, diabetic nephropathy, diabeticneuropathy, diabetic retinopathy), influenza, asthma, psoriasis,glomerulonephritis, septic shock, and ulcerative colitis.

Molecules of the invention are useful in promoting angiogenesis andwound healing (for example, wounds, burns, and bone fractures). They arealso useful as an adjuvant to enhance immune responsiveness to specificantigen and/or anti-viral immune responses.

More generally, the molecules of the invention are useful in modulatingthe immune response. For example, they may be useful in preparing for orrecovering from surgery, trauma, radiation therapy; chemotherapy, andtransplantation, or may be used to boost the immune response and/or therecovery process in elderly and immunocompromised individuals. They areuseful as immunosuppressive agents, for example, in the treatment orprevention of autoimmune disorders. In specific embodiments, moleculesof the invention are used to treat or prevent chronic inflammatory,allergic, or autoimmune conditions, such as those described herein orotherwise known in the art.

The uses of the molecules of the invention include, but are not limitedto, the treatment or prevention of adult respiratory distress syndrome,anaphylaxis, allergic asthma, allergen rhinitis, drug allergies (forexample, to penicillin or cephalosporins), primary central nervoussystem lymphoma (PCNSL), glioblastoma, chronic lymphocytic leukemia(CLL), lymphadenopathy, rheumatoid arthritis, osteoarthritis, acutelymphoblastic leukemia (ALL), Hodgkin's disease and non-Hodgkin'slymphoma, opthalmopathy, uveoretinitis, the autoimmune phase of Type 1diabetes, myasthenia gravis, autoimmune hepatological disorder,autoimmune inflammatory bowel disease, and Crohn's disease. Thecombination of MGD-CSF protein with an immunotherapeutic agent such asIL-2 or IL-12 may result in synergistic or additive effects useful intreating established cancers.

Additionally, the molecules of the invention may be employed not only astherapeutic molecules as described herein, but additionally as researchtools in elucidating the biology of tumor-related diseases, such ascancer. Thus, molecules of the invention are useful for inhibiting themultiplication of a tumor cell or cancer cell, or for treating cancer inan animal. The molecules of the invention can be used accordingly in avariety of settings for the treatment of animal cancers such assarcomas, adenomas, adenocarcinomas, carcinomas, papillomas, lymphomas,and the like. Other particular types of cancers that can be treated withmolecules of the invention include, but are not limited to prostate,breast (including, for example, intraductal and inflammatory), colon,colorectal, bladder, ovarian, cervical, papillary carcinoma, papillaryadenocarcinomas, cystadenocarcinoma, embryonal carcinoma, uterinecancer, and testicular cancer.

Antibodies and Vaccines

Antibodies

Antibodies specific to MGD-CSF or NP_(—)689669 are suitable for use inthe present invention and can be raised against the intact MGD-CSFprotein or an antigenic polypeptide fragment thereof. The protein orfragment may be presented with or without a carrier protein, such as analbumin, to an animal, such as a rabbit or mouse). In general,polypeptide fragments are sufficiently immunogenic to produce asatisfactory immune response without a carrier if they are at leastabout 25 amino acids in length.

Antibodies of the invention include polyclonal and monoclonal antibodypreparations, as well as preparations including hybrid antibodies,altered antibodies, chimeric antibodies and, humanized antibodies, aswell as hybrid (chimeric) antibody molecules (see, for example, Winteret al., Nature 349:293-299 (1991)); and U.S. Pat. No. 4,816,567);F(ab′)₂ and F(ab) fragments; Fv molecules (noncovalent heterodimers,see, for example, Inbar et al., Proc. Natl. Acad. Sci. 69:2659-2662(1972)); and Ehrlich et al. (1980) Biochem 19:4091-4096); single chainFv molecules (sFv) (see, e.g., Huston et al., Proc. Natl. Acad. Sci.85:5879-5883 (1980)); dimeric and trimeric antibody fragment constructs;minibodies (see, e.g., Pack et al., Biochem. 31:1579-1584 (1992); Cumberet al., J. Immunology 149B:120-126 (1992)); humanized antibody molecules(see, e.g., Riechmann et al., Nature 332:323-327 (1988); Verhoeyan etal., Science 239:1534-1536 (1988)); heteroconjugate and bispecificantibodies (see, e.g., U.S. Pat. No. 6,010,902 and U.S. Patent Appln.2002/0155604); and any functional fragments obtained from suchmolecules, wherein such fragments retain specific binding.

Methods of making monoclonal and polyclonal antibodies are known in theart. Monoclonal antibodies are generally antibodies having a homogeneousantibody population. The term is not limited regarding the species orsource of the antibody, nor is it intended to be limited by the mannerin which it is made. The term encompasses whole immunoglobulins.Polyclonal antibodies are generated by immunizing a suitable animal,such as a mouse, rat, rabbit, sheep or goat, with an antigen ofinterest, such as a stem cell transformed with a gene encoding anantigen. In order to enhance immunogenicity, the antigen can be linkedto a carrier prior to immunization. Suitable carriers are typicallylarge, slowly metabolized macromolecules such as proteins,polysaccharides, polylactic acids, polyglycolic acids, polymeric aminoacids, amino acid copolymers, lipid aggregates (such as oil droplets orliposomes), and inactive virus particles. Such carriers are well knownto those of ordinary skill in the art. Furthermore, the antigen may beconjugated to a bacterial toxoid, such as a toxoid from diphtheria,tetanus, cholera, etc., in order to enhance the immunogenicity thereof.

In addition, techniques developed for the production of chimericantibodies (Morrison et al., Proc. Natl. Acad. Sci., 81:851-855 (1984);Neuberger et al., Nature, 312:604-608 (1984); Takeda et al., Nature,314:452-454 (1985)) by splicing genes from a mouse antibody molecule ofappropriate antigen specificity together with genes from a humanantibody molecule of appropriate biological activity can be used.Chimeric antibodies, which are antibodies in which different portionsare derived from different animal species, such as those having avariable region derived from a murine monoclonal antibody and a humanimmunoglobulin constant region, for example, humanized antibodies, andinsertion/deletions relating to cdr and framework regions, are suitablefor use in the invention.

The invention also includes humanized antibodies, i.e., those withmostly human immunoglobulin sequences. Humanized antibodies of theinvention generally refer to non-human immunoglobulins that have beenmodified to incorporate portions of human sequences. A humanizedantibody may include a human antibody that contains entirely humanimmunoglobulin sequences.

The antibodies of the invention may be prepared by any of a variety ofmethods. For example, cells expressing an MGD-CSF or NP-689669 proteinor an antigenic fragment thereof can be administered to an animal inorder to induce the production of sera containing polyclonal antibodies.A preparation of MGD-CSF or NP_(—)689669 protein can be prepared andpurified to render it substantially free of natural contaminants, andthe preparation introduced into an animal in order to produce polyclonalantisera with specific binding activity.

Antibodies of the invention specifically bind to their respectiveantigen(s); they may display high avidity and/or high affinity to aspecific polypeptide, or more accurately, to an epitope of an antigen.Antibodies of the invention may bind to one epitope, or to more than oneepitope. They may display different affinities and/or avidities todifferent epitopes on one or more molecules. When an antibody binds morestrongly to one epitope than to another, adjusting the bindingconditions can, in some instances, result in antibody binding almostexclusively to the specific epitope and not to any other epitopes on thesame polypeptide, and not to a polypeptide that does not comprise theepitope.

The invention also provides monoclonal antibodies and MGD-CSF orNP_(—)689669 protein binding fragments thereof. Monoclonal antibodies ofthe invention can be prepared using hybridoma technology, for example,Kohler et al., Nature, 256:495 (1975); Kohler et al., Eur. J. Immunol.,6:511 (1976); Kohler et. al., Eur. J. Immunol., 6:292 (1976); Hammerlinget al., in: Monoclonal Antibodies and T-Cell Hybridomas, Elsevier, N.Y.,(1981) pp. 563-681. In general, such procedures involve immunizing ananimal (for example, a mouse) with an MGD-CSF protein antigen or with anMGD-CSF protein-expressing cell. Suitable cells can be recognized bytheir capacity to bind anti-MGD-CSF protein antibody. Such cells may becultured in any suitable tissue culture medium; for example, in Earle'smodified Eagle's medium supplemented with 10% fetal bovine serum(inactivated at about 56° C.), and supplemented with about 10grams/liter of nonessential amino acids, about 1,000 U/ml of penicillin,and about 100 μg/ml of streptomycin. The splenocytes of such mice areextracted and fused with a suitable myeloma cell line. Any suitablemyeloma cell line may be employed in accordance with the presentinvention; e.g., the parent myeloma cell line (SP20), available from theAmerican Type Culture Collection (ATCC), Manassas, Va. After fusion, theresulting hybridoma cells are selectively maintained in HAT medium, andthen cloned by limiting dilution, for example, as described by Wands etal., Gastroenterology, 80:225-232 (1981).

Alternatively, antibodies capable of binding to the MGD-CSF orNP_(—)689669 protein antigen may be produced in a two-step procedurethrough the use of anti-idiotypic antibodies. Such a method makes use ofthe fact that antibodies are themselves antigens, and that, therefore,it is possible to obtain an antibody which binds to a second antibody.In accordance with this method, specific antibodies are used to immunizean animal such as a mouse. The splenocytes of such an animal are thenused to produce hybridoma cells, and the hybridoma cells are screened toidentify clones which produce an antibody whose ability to bind to thespecific antibody can be blocked by the antigen. Such antibodiescomprise anti-idiotypic antibodies to the MGD-CSF or NP_(—)689669protein-specific antibody and can be used to immunize an animal toinduce formation of further specific antibodies.

It will be appreciated that Fab and F(ab′)₂ and other fragments of theantibodies of the present invention may be used according to the methodsdisclosed herein. Such fragments are typically produced by proteolyticcleavage, using enzymes such as papain (to produce Fab fragments) orpepsin (to produce F(ab′)₂ fragments). Alternatively, MGD-CSFprotein-binding fragments can be produced through the application ofrecombinant DNA technology or through synthetic chemistry. Humanizedchimeric monoclonal antibodies are suitable for in vivo use ofanti-MGD-CSF in humans. Such humanized antibodies can be produced usinggenetic constructs derived from hybridoma cells producing the monoclonalantibodies described above. Methods for producing chimeric antibodiesare known in the art. See, for review, Morrison, Science, 229:1202(1985); Oi et al., BioTechniques, 4:214 (1986); Cabilly et al., U.S.Pat. No. 4,816,567; Taniguchi et al., EP 0 171 496; Morrison et al., EP0 173 494; Neuberger et al., WO 8601533; Robinson et al., WO 8702671;Boulianne et al., Nature, 312:643 (1984); Neuberger et al., Nature,314:268 (1985).

Vaccines

The invention provides a method for prophylactic or therapeutictreatment of a subject needing or desiring such treatment by providing avaccine, that can be administered to the subject. It also provides amethod for enhancing immune response to a subject by providing asubstantially purified polypeptide from SEQ. ID. NOS.:7-12 or an activefragment; providing a vaccine composition, and administering thepolypeptide and vaccine compositions to the subject. The vaccine maycomprise one or more of a polynucleotide, polypeptide, or modulator ofthe invention, for example an antibody vaccine composition, apolypeptide vaccine composition, or a polynucleotide vaccinecomposition, useful for treating cancer, proliferative, inflammatory,immune, metabolic, bacterial, or viral disorders.

For example, the vaccine can be a cancer vaccine, and the polypeptidecan concomitantly be a cancer antigen. The vaccine may be ananti-inflammatory vaccine, and the polypeptide can concomitantly be aninflammation-related antigen. The vaccine may be a viral vaccine, andthe polypeptide can concomitantly be a viral antigen. In someembodiments, the vaccine comprises a polypeptide fragment, comprising atleast one extracellular fragment of a polypeptide of the invention,and/or at least one extracellular fragment of a polypeptide of theinvention minus the signal peptide, for the treatment, for example, ofproliferative disorders, such as cancer. In certain embodiments, thevaccine comprises a polynucleotide encoding one or more such fragments,administered for the treatment, for example, of proliferative disorders,such as cancer. Further, the vaccine can be administered with or withoutan adjuvant. The vaccine can be administered with polypeptides shown inthe Tables and Sequence Listing; it may be administered prior to,substantially contemporaneously with, or after administering thepolypeptides.

Vaccine therapy involves the use of polynucleotides, polypeptides, oragents of the invention as immunogens for tumor antigens (Machiels etal., Semin. Oncol. 29: 494-502, 2002). For example, peptide-basedvaccines of the invention include unmodified subject polypeptides,fragments thereof, and MHC class I and class II-restricted peptide(Knutson et al., J. Clin. Invest. 07:477-484, 2001), comprising, forexample, the disclosed sequences with universal, nonspecific MHC classII-restricted epitopes. Peptide-based vaccines comprising a tumorantigen can be given directly, either alone or in conjunction with othermolecules. The vaccines can also be delivered orally by producing theantigens in transgenic plants that can be subsequently ingested (U.S.Pat. No. 6,395,964).

In some embodiments, antibodies themselves can be used as antigens inanti-idiotype vaccines. That is, administering an antibody to a tumorantigen stimulates B cells to make antibodies to that antibody, which inturn recognize the tumor cells

Nucleic acid-based vaccines can deliver tumor antigens as polynucleotideconstructs encoding the antigen. Vaccines comprising genetic material,such as DNA or RNA, can be given directly, either alone or inconjunction with other molecules. Administration of a vaccine expressinga molecule of the invention, e.g., as plasmid DNA, leads to persistentexpression and release of the therapeutic immunogen over a period oftime, helping to control unwanted tumor growth.

In some embodiments, nucleic acid-based vaccines encode subjectantibodies. In such embodiments, the vaccines (e.g., DNA vaccines) caninclude post-transcriptional regulatory elements, such as thepost-transcriptional regulatory acting RNA element (WPRE) derived fromWoodchuck Hepatitis Virus. These post-transcriptional regulatoryelements can be used to target the antibody, or a fusion proteincomprising the antibody and a co-stimulatory molecule, to the tumormicroenvironment (Pertl et al., Blood, 101:649-654, 2003).

Besides stimulating anti-tumor immune responses by inducing humoralresponses, vaccines of the invention can also induce cellular responses,including stimulating T-cells that recognize and kill tumor cellsdirectly. For example, nucleotide-based vaccines of the inventionencoding tumor antigens can be used to activate the CD8⁺ cytotoxic Tlymphocyte arm of the immune system.

In some embodiments, the vaccines activate T-cells directly, and inothers they enlist antigen-presenting cells to activate T-cells. KillerT-cells are primed, in part, by interacting with antigen-presentingcells, for example, dendritic cells. In some embodiments, plasmidscomprising the nucleic acid molecules of the invention enterantigen-presenting cells, which in turn display the encodedtumor-antigens that contribute to killer T-cell activation. Again, thetumor antigens can be delivered as plasmid DNA constructs, either aloneor with other molecules.

Since MGD-CSF and NP_(—)689669 can promote dendritic celldifferentiation in vitro from either human bone marrow CD34⁺ stem cellsor peripheral blood monocytes, molecules of the invention can be used toexpand dendritic cells ex vivo. The expanded cell population can then bereturned to the patient, for example, as a dendritic cell vaccine.Furthermore, molecules of the invention may promote dendritic celldifferentiation from autologous hematopoietic stem cells and/ormonocytes in vivo in the patient, which will enhance the patient'santigen presenting capability, and contribute to the ability to combatcertain diseases, such as cancer.

In further embodiments, RNA can be used in vaccine production. Forexample, dendritic cells can be transfected with RNA encoding tumorantigens (Heiser et al., J. Clin. Invest. 109:409-417, 2002; Mitchelland Nair, J. Clin. Invest. 106: 1065-1069, 2000). This approachovercomes the limitations of obtaining sufficient quantities of tumormaterial, extending therapy to patients otherwise excluded from clinicaltrials. For example, a subject RNA molecule isolated from tumors can beamplified using RT-PCR. In some embodiments, the RNA molecule of theinvention is directly isolated from tumors and transfected intodendritic cells with no intervening cloning steps.

In some embodiments the molecules of the invention are altered such thatthe peptide antigens are more highly antigenic than in their nativestate. These embodiments address the need in the art to overcome thepoor in vivo immunogenicity of most tumor antigens by enhancing tumorantigen immunogenicity via modification of epitope sequences (Yu andRestifo, J. Clin. Invest. 110:289-294, 2002).

Another recognized problem of cancer vaccines is the presence ofpreexisting neutralizing antibodies. Some embodiments of the presentinvention overcome this problem by using viral vectors fromnon-mammalian natural hosts, for example, avian pox viruses. Alternativeembodiments that also circumvent preexisting neutralizing antibodiesinclude genetically engineered influenza viruses, and the use of nakedplasmid DNA vaccines that contain DNA with no associated protein (Yu andRestifo, J. Clin. Invest. 110:289-294, 2002).

All of the immunogenic methods of the invention can be used alone or incombination with other conventional or unconventional therapies. Forexample, immunogenic molecules can be combined with other molecules thathave a variety of antiproliferative effects, or with additionalsubstances that help stimulate the immune response, i.e., adjuvants orcytokines.

For example, in some embodiments, nucleic acid vaccines encode analphaviral replicase enzyme, in addition to tumor antigens. Thisapproach to vaccine therapy successfully combines therapeutic antigenproduction with the induction of the apoptotic death of the tumor cell(Yu and Restifo, J. Clin. Invest. 110:289-294, 2002).

In some embodiments, a molecule of the invention is involved in thecontrol of cell proliferation, and an agent of the invention inhibitsundesirable cell proliferation. Such agents are useful for treatingdisorders that involve abnormal cell proliferation, including, but notlimited to, cancer, psoriasis, and scleroderma. Whether a particularagent and/or therapeutic regimen of the invention is effective inreducing unwanted cellular proliferation, e.g., in the context oftreating cancer, can be determined using standard methods. For example,the number of cancer cells in a biological sample (e.g., blood, a biopsysample, and the like), can be determined. The tumor mass can bedetermined using standard radiological or biochemical methods.

The molecules of the invention find use in immunotherapy ofhyperproliferative disorders, including cancer, neoplastic, andparaneoplastic disorders. That is, the subject molecules can correspondto tumor antigens, of which over 1770 have been identified to date (Yuand Restifo, J. Clin. Invest. 110:289-294, 2002). Immunotherapeuticapproaches include passive immunotherapy and vaccine therapy and canaccomplish both generic and antigen-specific cancer immunotherapy.

Passive immunity approaches involve antibodies of the invention that aredirected toward specific tumor-associated antigens. Such antibodies caneradicate systemic tumors at multiple sites, without eradicating normalcells. In some embodiments, the antibodies are combined with radioactivecomponents, as provided above, for example, combining the antibody'sability to specifically target tumors with the added lethality of theradioisotope to the tumor DNA.

Useful antibodies comprise a discrete epitope or a combination of nestedepitopes, i.e., a 10-mer epitope and associated peptide multimersincorporating all potential 8-mers and 9-mers, or overlapping epitopes(Dutoit et al., J. Clin. Invest. 110:1813-1822, 2002). Thus a singleantibody can interact with one or more epitopes. Further, the antibodycan be used alone or in combination with different antibodies, that allrecognize either a single or multiple epitopes.

Neutralizing antibodies can provide therapy for cancer and proliferativedisorders. Neutralizing antibodies that specifically recognize a proteinor peptide of the invention can bind to the protein or peptide, e.g., ina bodily fluid or the extracellular space, thereby modulating thebiological activity of the protein or peptide. For example, neutralizingantibodies specific for proteins or peptides that play a role instimulating the growth of cancer cells can be useful in modulating thegrowth of cancer cells. Similarly, neutralizing antibodies specific forproteins or peptides that play a role in the differentiation of cancercells can be useful in modulating the differentiation of cancer cells.

MGD-CSF “Knock-Outs” and Homologous Recombination

Endogenous gene expression can be reduced by inactivating or “knockingout” a gene of interest and/or its promoter using targeted homologousrecombination, for example, Smithies et al., Nature, 317:230-234 (1985);Thomas et al., Cell, 51:503-512 (1987); and Thompson et al., Cell,5:313-321 (1989). For example, a mutant, non-functional polynucleotideof the invention (or a completely unrelated DNA sequence) flanked by DNAhomologous to the endogenous polynucleotide sequence (either the codingregions or regulatory regions of the gene) can be used, with or withouta selectable marker and/or a negative selectable marker, to transfectcells that express polypeptides of the invention in vivo. In anotherembodiment, techniques known in the art are used to generate knockoutsin cells that contain, but do not express, the gene of interest.Insertion of the DNA construct, via targeted homologous recombination,results in inactivation of the targeted gene. Such approaches areparticularly suited in research and agricultural fields wheremodifications to embryonic stem cells can be used to generate animaloffspring with an inactive targeted gene, for example, Thomas et al.,Cell 51:503-512, (1987); Thompson (1989), supra). However, this approachcan be routinely adapted for use in humans provided the recombinant DNAconstructs are directly administered or targeted to the required site invivo using appropriate viral vectors that will be apparent to those ofskill in the art.

In further embodiments of the invention, cells that are geneticallyengineered to express the polypeptides of the invention, oralternatively, that are genetically engineered not to express thepolypeptides of the invention, such as knockouts, are administered to apatient in vivo. Such cells may be obtained from the patient, includinghumans and non-human animals, or an MHC compatible donor, and caninclude, but are not limited to, fibroblasts, bone marrow cells, bloodcells (for example, lymphocytes), adipocytes, muscle cells, endothelialcells, etc. The cells are genetically engineered in vitro usingrecombinant DNA techniques to introduce the coding sequence ofpolypeptides of the invention into the cells, or alternatively, todisrupt the coding sequence and/or endogenous regulatory sequenceassociated with the polypeptides of the invention, e.g., by transduction(using viral vectors, and/or vectors that integrate the transgene intothe cell genome) or transfection procedures, including, but not limitedto, the use of plasmids, cosmids, YACs, naked DNA, electroporation,liposomes, etc. The coding sequence of the polypeptides of the inventioncan be placed under the control of a strong constitutive or induciblepromoter or promoter/enhancer to achieve expression, and secretion, ofthe polypeptides of the invention. The engineered cells which expressand secrete the polypeptides of the invention can be introduced into thepatient systemically, e.g., in the circulation, or intraperitoneally.Alternatively, the cells can be incorporated into a matrix and implantedin the body, e.g., genetically engineered fibroblasts can be implantedas part of a skin graft; genetically engineered endothelial cells can beimplanted as part of a lymphatic or vascular graft. (See, for example,Anderson et al. U.S. Pat. No. 5,399,349; and Mulligan & Wilson, U.S.Pat. No. 5,460,959).

When the cells to be administered are non-autologous or non-MHCcompatible cells, they can be administered using well known techniqueswhich prevent the development of a host immune response against theintroduced cells. For example, the cells may be introduced in anencapsulated form which, while allowing for an exchange of componentswith the immediate extracellular environment, does not allow theintroduced cells to be recognized by the host immune system.

Transgenic Non-Human Animals

The polypeptides of the invention can also be expressed in transgenicnon-human animals. Animals of any species, including, but not limitedto, mice, rats, rabbits, hamsters, guinea pigs, pigs, micro-pigs, goats,sheep, cows, and non-human primates, for example, baboons, monkeys, andchimpanzees may be used to generate transgenic animals. In a specificembodiment, techniques described herein or otherwise known in the art,are used to express polypeptides of the invention in humans, as part ofa gene therapy protocol.

Any technique known in the art may be used to introduce the transgene(embodied in polynucleotides shown in the Sequence Listing) into animalsto produce a founder lines of transgenic animals. Such techniquesinclude, but are not limited to, pronuclear microinjection (Paterson etal., Appl. Microbiol. Biotechnol. 40:691-698 (1994); Carver et al.,Biotechnology (NY) 11:1263-1270 (1993); Wright et al., Biotechnology(NY) 9:830-834 (1991); and Hoppe et al., U.S. Pat. No. 4,873,191(1989)); retrovirus mediated gene transfer into germ lines (Van derPutten et al., Proc. Natl. Acad. Sci. 82:6148-6152 (1985)), blastocystsor embryos; gene targeting in embryonic stem cells (Thompson et al.,Cell 56:313-321 (1989)); electroporation of cells or embryos (Lo, Mol.Cell. Biol. 3:1803-1814 (1983)); introduction of the polynucleotides ofthe invention using a gene gun (see, for example, Ulmer et al., Science259:1745 (1993); introducing nucleic acid constructs into embryonicpluripotent stem cells and transferring the stem cells back into theblastocyst; and sperm-mediated gene transfer (Lavitrano et al., Cell57:717-723 (1989); etc. For a review of such techniques, see Gordon,Intl. Rev. Cytol. 115:171-229 (1989). See also, U.S. Pat. No. 5,464,764(Capecchi et al., Positive-Negative Selection Methods and Vectors); U.S.Pat. No. 5,631,153 (Capecchi et al., Cells and Non-Human OrganismsContaining Predetermined Genomic Modifications and Positive-NegativeSelection Methods and Vectors for Making Same); U.S. Pat. No. 4,736,866(Leder et al., Transgenic Non-Human Animals); and U.S. Pat. No.4,873,191 (Wagner et al., Genetic Transformation of Zygotes). Anytechnique known in the art may be used to produce transgenic clonescontaining polynucleotides of the invention, for example, nucleartransfer into enucleated oocytes of nuclei from cultured embryonic,fetal, or adult cells induced to quiescence (Campbell et al., Nature380:64-66 (1996); Wilmut et al., Nature 385:810-813 (1997)).

The invention provides for transgenic animals that carry the transgenein all their cells, as well as animals which carry the transgene insome, but not all their cells, such as mosaic or chimeric animals. Thetransgene may be integrated as a single transgene or as multiple copiessuch as in concatamers, for example, head-to-head tandems orhead-to-tail tandems. The transgene may also be selectively introducedinto and activated in a particular cell type by following, for example,the teaching of Lasko et al. (Proc. Natl. Acad. Sci. 89:6232-6236(1992)). The regulatory sequences required for such a cell-type specificactivation will depend upon the particular cell type of interest, andwill be apparent to those of skill in the art. It may be desired thatthe polynucleotide transgene be integrated into the chromosomal site ofthe endogenous gene, gene targeting is then suitable. Briefly, when sucha technique is to be utilized, vectors containing some nucleotidesequences homologous to the endogenous gene are designed for the purposeof integrating, via homologous recombination with chromosomal sequences,into and disrupting the function of the nucleotide sequence of theendogenous gene. The transgene may also be selectively introduced into aparticular cell type, thus inactivating the endogenous gene in only thatcell type, by following, for example, the teaching of Gu et al. (Science265:103-106 (1994)). The regulatory sequences required for such acell-type specific inactivation will depend upon the particular celltype of interest, and will be apparent to those of skill in the art.

Once transgenic animals have been generated, the expression of therecombinant gene may be assayed using standard techniques. Initialscreening may be accomplished by Southern blot analysis or PCRtechniques to analyze animal tissues to verify that integration of thetransgene has taken place. The level of mRNA expression of the transgenein the tissues of the transgenic animals may also be assessed usingtechniques which include, but are not limited to, Northern blot analysisof tissue samples obtained from the animal, in situ hybridizationanalysis, and reverse transcriptase-PCR (RT-PCR). Samples of transgenicgene-expressing tissue may also be evaluated immunocytochemically orimmunohistochemically using antibodies specific for the transgeneproduct.

Once the founder animals are produced, they may be bred, inbred,outbred, or crossbred to produce colonies of the particular animal.Examples of such breeding strategies include, but are not limited tooutbreeding of founder animals with more than one integration site inorder to establish separate lines; inbreeding of separate lines in orderto produce compound transgenics that express the transgene at higherlevels because of the effects of additive expression of each transgene;crossing of heterozygous transgenic animals to produce animalshomozygous for a given integration site in order to both augmentexpression and eliminate the need for screening of animals by DNAanalysis; crossing of separate homozygous lines to produce compoundheterozygous or homozygous lines; and breeding to place the transgene ona distinct background that is appropriate for an experimental model ofinterest.

Transgenic and “knock-out” animals of the invention have uses whichinclude, but are not limited to, animal model systems useful inelaborating the biological function of molecules of the inventionstudying conditions and/or disorders associated with aberrant expressionof molecules of the invention, and in screening for compounds effectivein ameliorating such conditions and/or disorders.

Kits

The present invention provides kits that can be used in the abovemethods. In one embodiment, a kit comprises an antibody of theinvention, for example, a purified antibody, in one or more containers.In an embodiment, the kits of the invention contain a substantiallyisolated polypeptide comprising an epitope which is specificallyimmunoreactive with an antibody included in the kit. The kits of theinvention may also comprise a control antibody which does not react withthe polypeptide of interest.

In an embodiment, the kits of the present invention comprise a means fordetecting the binding of an antibody to a polypeptide of interest. Forexample, the antibody may be conjugated to a detectable substrate suchas a fluorescent compound, an enzymatic substrate, a radioactivecompound or a luminescent compound, or a second antibody whichrecognizes the first antibody may be conjugated to a detectablesubstrate).

In an embodiment, the kit is a diagnostic kit for use in screening serumcontaining antibodies specific against MGD-CSF related molecules. Such akit may include a control antibody that does not react with thepolypeptide of interest. Such a kit may include a substantially isolatedpolypeptide antigen comprising an epitope which is specificallyimmunoreactive with at least one anti-polypeptide antigen antibody.Further, such a kit includes means for detecting the binding of theantibody to the antigen. The antibody may be conjugated to a fluorescentcompound, such as fluorescein or rhodamine, which can be detected byflow cytometry. In an embodiment, the kit may include a recombinantlyproduced or chemically synthesized polypeptide antigen. The polypeptideantigen of the kit may also be attached to a solid support.

In a further embodiment, the detecting means of the above-described kitincludes a solid support to which said polypeptide antigen is attached.Such a kit may also include a non-attached reporter-labeled anti-humanantibody. In this embodiment, binding of the antibody to the polypeptideantigen can be detected by binding of the said reporter-labeledantibody.

In an additional embodiment, the invention includes a diagnostic kit foruse in screening serum containing antigens of the polypeptide of theinvention. The diagnostic kit includes a substantially isolated antibodyspecifically immunoreactive with polypeptide or polynucleotide antigens,and means for detecting the binding of the polynucleotide or polypeptideantigen to the antibody. In an embodiment, the antibody is attached to asolid support. In an embodiment, the antibody is a monoclonal antibody.The detecting means of the kit may include a second, labeled monoclonalantibody. Alternatively, or in addition, the detecting means may includea labeled, competing antigen.

In a diagnostic configuration, test serum is reacted with a solid phasereagent having a surface-bound antigen obtained by the methods of thepresent invention. After binding with specific antigen antibody to thereagent and removing unbound serum components by washing, the reagent isreacted with reporter-labeled anti-human antibody to bind reporter tothe reagent in proportion to the amount of bound anti-antigen antibodyon the solid support. The reagent is again washed to remove unboundlabeled antibody, and the amount of reporter associated with the reagentis determined. Typically, the reporter is an enzyme which is detected byincubating the solid phase in the presence of a suitable fluorometric,luminescent or colorimetric substrate.

The solid surface reagent may be prepared by known techniques forattaching protein material to solid support material, such as polymericbeads, dip sticks, 96-well plates, and/or filter material. Theseattachment methods generally include non-specific adsorption of theprotein to the support or covalent attachment of the protein, typicallythrough a free amine group, to a chemically reactive group on the solidsupport, such as an activated carboxyl, hydroxyl, or aldehyde group.Alternatively, streptavidin coated plates can be used in conjunctionwith a biotinylated antigen.

Additional objects and advantages of the invention will be set forth inpart in the description which follows, and in part will be obvious fromthe description, or may be learned by practice of the invention. Theobjects and advantages of the invention will be realized and attained bymeans of the elements and combinations particularly pointed out in theappended claims. Moreover, advantages described in the body of thespecification, if not included in the claims, are not per se limitationsto the claimed invention.

It is to be understood that both the foregoing general description andthe following detailed description are exemplary and explanatory onlyand are not restrictive of the invention, as claimed. Moreover, it mustbe understood that the invention is not limited to the particularembodiments described, as such may, of course, vary. Further, theterminology used to describe particular embodiments is not intended tobe limiting, since the scope of the present invention will be limitedonly by its claims. The claims do not encompass embodiments in thepublic domain.

With respect to ranges of values, the invention encompasses eachintervening value between the upper and lower limits of the range to atleast a tenth of the lower limit's unit, unless the context clearlyindicates otherwise. Further, the invention encompasses any other statedintervening values. Moreover, the invention also encompasses rangesexcluding either or both of the upper and lower limits of the range,unless specifically excluded from the stated range.

Unless defined otherwise, the meanings of all technical and scientificterms used herein are those commonly understood by one of ordinary skillin the art to which this invention belongs. One of ordinary skill in theart will also appreciate that any methods and materials similar orequivalent to those described herein can also be used to practice ortest the invention. Further, all publications mentioned herein areincorporated by reference in their entireties.

It must be noted that, as used herein and in the appended claims, thesingular forms “a,” “or,” and “the” include plural referents unless thecontext clearly dictates otherwise. Thus, for example, reference to “asubject polypeptide” includes a plurality of such polypeptides andreference to “the agent” includes reference to one or more agents andequivalents thereof known to those skilled in the art, and so forth.

Further, all numbers expressing quantities of ingredients, reactionconditions, % purity, polypeptide and polynucleotide lengths, and soforth, used in the specification and claims, are modified by the term“about,” unless otherwise indicated. Accordingly, the numericalparameters set forth in the specification and claims are approximationsthat may vary depending upon the desired properties of the presentinvention. At the very least, and not as an attempt to limit theapplication of the doctrine of equivalents to the scope of the claims,each numerical parameter should at least be construed in light of thenumber of reported significant digits, applying ordinary roundingtechniques. Nonetheless, the numerical values set forth in the specificexamples are reported as precisely as possible. Any numerical value,however, inherently contains certain errors from the standard deviationof its experimental measurement.

The specification is most thoroughly understood in light of thereferences cited herein. Each of these references is hereby incorporatedby the reference in its entirety.

EXAMPLES

The examples, which are intended to be exemplary of the invention andshould therefore not be considered to limit the invention in any way,also describe and provide detail for aspects and embodiments of theinvention discussed above. The examples are not intended to representthat the experiments below are all or the only experiments performed.Efforts have been made to ensure accuracy with respect to numbers used(for example, amounts, temperature, etc.) but some experimental errorsand deviations should be accounted for. Unless indicated otherwise,parts are parts by weight, molecular weight is weight average molecularweight, temperature is in degrees Centigrade, and pressure is at or nearatmospheric.

Example 1 Amino Acid Sequence Alignment of MGD-CSF with MCG34647

MGD-CSF and NP_(—)689669 were compared by aligning their amino acidsusing the program clustal format for T-COFFEE Version 1.37, cpu=0.00sec., score=72, Nseq=3, len=242. As shown in FIG. 1, the amino acidsequence of MGD-CSF differs from the amino acid sequence of NP_(—)689669(MCG34647). The latter sequence has a glutamine (Q) residue at aminoacid 81. The five flanking amino acid residues adjacent to and on eitherside of amino acid 81 in the NCBI sequence of MCG34647 are NVTRLQRAQVS(SEQ ID NO.:279). In contrast to this published sequence of MCG34647,MGD-CSF contains the amino acid sequence NVTRLRAQVS (SEQ ID NO.:280).The difference between these sequences results from alternative splicingof the MCG34647 gene between exons 3 and 4. The genome sequences at theexon 3-4 boundary are the codons aac gtc acc agg ctg gtg (SEQ IDNO.:281) and cag cag agg gcc cag gtg agc (SEQ ID NO.:282), wherein thegtg codon (shown in italics) represents the 5′ splice donor site at theend of exon 3, and the two cag codons (shown in italics) represent twoalternative splice acceptor sites at the beginning of exon 4. Thus, thepublished MCG34647 sequence represents a transcript resulting from theuse of the first cag splice acceptor site, while the MGD-CSF sequencerepresents a transcript resulting from the use of the second cag spliceacceptor site.

The MCG34647 glutamine 81 residue is encoded by the second cag codon,which is not spliced out when the first cag splice acceptor site isused. In contrast, the use of the second cag as the splice acceptor siteresults in the first cag sequence being spliced out of the resulting RNAtranscript, which in turn results in the lack of a correspondingglutamine in the MGD-CSF splice variant. Hence, MGD-CSF is a splicevariant and represents RNA and protein species that are distinct fromMCG34647.

Example 2 Plasmid Vectors for MGD-CSF Expression

The MGD-CSF gene was cloned into pTT-5 and pTT-2 mammalian expressionvectors modified as shown in FIG. 2 and FIG. 3 using standard cloningprocedures. The MGD-CSF gene was also cloned into the pIB/V5His-DESTinsect cell expression vector (Invitrogen, Carlsbad Calif.) usingstandard cloning procedures. The resulting constructs are described inTable 1 and Table 5. They include human MGD-CSF untagged in vector pTT5(MGD-CSF), human MGD-CSF untagged in vector pTT2 (CLN00839395), humanMGD-CSF with a C-terminal V5H8 tag in vector pTT5 (CLN00732663), humanMGD-CSF tagged with V5H8 (CLN00732663), human MGD-CSF with a C-terminalV5H8 tag in vector pTT2 (CLN00840351), human MGD-CSF with a C-terminalV5H8 tag in vector pIB/V5His-DEST, (CLN00758593), and human MGD-CSF witha collagen secretory leader and a C-terminal Streptag in vector pTT5-G(CLN00816424).

To monitor the expression and secretion of MGD-CSF and to aid in itspurification, the construct CLN00821867 was generated with a TobaccoEtch Virus (TEV) protease recognition site engineered between theprotein and a C-terminal cleavable tag. The seven amino acid recognitionsite for TEV protease is Glu-Asn-Leu-Tyr-Phe-Gln-Gly (SEQ ID NO.:283)with cleavage occurring between Gln and Gly. Construct CLN00821867 wasdesignated MGD-CSF(1 to 241aa)_TEV_V5_Streptag II_H8 and C-tagged invector pTT5-I.

To improve the secretion of MGD-CSF, its secretory signal peptide, whichis encoded by the first 20 amino acids, was replaced by the 23 aminoacids that encode the signal peptide of collagen (GenBank proteinaccession number NP_(—)001842). This construct, CLN00848149, wasdesignated MGD-CSF collagen SP(1-23aa)_MGD-CSF(21 to 241aa), and isuntagged in vector pTT5-G. Another such construct also has a TEVprotease recognition site engineered between the protein and theC-terminal cleavable tag. CLN00816424 was designated MGD-CSF collagenSP(1-23aa)_MGD-CSF(21 to 241aa)_TEV_V5_Streptag II_H8, and is C-taggedin vector pTT5-G. A third such construct was generated with a TEV siteengineered between the N-terminal tag and the protein. CLN00816425 wasdesignated MGD-CSF collagenSP(1-23aa)_H8_Streptag II_V5_TEV_MGD-CSF(21to 241aa), and is N-tagged in vector pTT5-H.

Deletion constructs were generated in which amino acids were deletedfrom the N-terminal, C-terminal, or both ends of mature proteins. TheMGD-CSF signal peptide of these deletion constructs was replaced withthe collagen signal peptide. CLN00848160 has 25 N-terminal amino acidsdeleted; it was designated MGD-CSF collagenSP(1-23aa)_MGD-CSF(26 to 241aa), and is untagged in vector pTT5. CLN00848173 has 30 N-terminal aminoacids deleted; it was designated MGD-CSF collagenSP(1-23aa)_MGD-CSF(31to 241aa), and is untagged in vector pTT5. CLN00848209 has 5 C-terminalamino acids and 20 N-terminal amino acids (signal peptide) deleted; itwas designated MGD-CSF collagenSP(1-23aa)_MGD-CSF(21 to 236aa), and isuntagged in vector pTT5. CLN00848197 has 10 C-terminal amino acids and20 N-terminal amino acids (signal peptide) deleted; it was designatedMGD-CSF collagenSP(1-23aa)_MGD-CSF(21 to 231aa), and is untagged invector pTT5. CLN00848185 has 28 C-terminal amino acids and 20 N-terminalamino acids (signal peptide) deleted; it was designated MGD-CSFcollagenSP(1-23aa)_MGD-CSF(21 to 213aa), and is untagged in vector pTT5.CLN00848220 has 25 N-terminal amino acids and 10 C-terminal amino acidsdeleted; it was designated MGD-CSF collagenSP(1-23aa)_MGD-CSF(26 to231aa), and is untagged in vector pTT5.

Two mouse orthologs of MGD-CSF were identified and cloned by standardprocedures into untagged pTT5 (CLN00840257 and CLN00847948) and intopTT5-I tagged with a TEV site between the clone and the tag (CLN00840253and CLN00842712). These orthologues can be used to perform animalstudies relating to the biological activity of MGD-CSF in mouse tissuesand cells.

The mouse orthologs represented by constructs CLN00840257 andCLN00840235, were derived from Mus musculus adult male small intestinecDNA clone 2010004A03 from the RIKEN full-length enriched library;hypothetical protein 12842043; at locus AK008082. The constructCLN00840257 represents the open reading frame (ORF) of the nucleotidesequence of phantom clone 2010004A03, and is a mouse ortholog of humanMGD-CSF cloned into vector pTT5. The construct CLN00840253 representsthe ORF of the nucleotide sequence of phantom clone 2010004A03, and wascloned into vector pTT5-L

The mouse orthologs represented by constructs CLN00847948 andCLN00842712 were derived from Mus musculus cDNA clone 2010004A03, mRNA(cDNA clone MGC:28891 IMAGE:4912097), complete cds 18921436, from theRIKEN full-length enriched library, at locus BC016254. The constructCLN00847948 (18921436) represents the ORF nucleotide sequence of humanMGD-CSF cloned into vector pTT5. The construct CLN00842712 (18921436)represents the ORF nucleotide sequence of human MGD-CSF cloned intovector pTT5.

Example 3 Transient Expression in Mammalian Cells

Complementary DNA encoding the MGD-CSF polypeptide was cloned into theexpression vectors pTT5 and pCDNA-pDEST40 and expressed as both a taggedand untagged protein. Protein levels were quantified by measuring thelevels of the tag, for example, a V5H is tag, by quantitative Westernblot analysis. The expression vectors were transfected into adherent293T cells using the transfection agent Fugene 6 (Roche, Nutley N.J.) inDMEM with 10% fetal bovine serum (FBS) and penicillin/streptomycin (100μg/ml, 100 U/ml), and incubated at 37° C. in 5% CO₂ for 40 hours, afterwhich the cells were washed with PBS and incubated for an additional 48hours in complete DMEM. Cell supernatant was harvested, cleared of celldebris by centrifugation, and tested for biological activity (untaggedcDNA) and protein expression (V5 tagged cDNA) by Western blot assayusing an anti-VS antibody.

Expression studies were also performed with 293-6E cells transientlyexpressing tagged MGD-CSF constructs in suspension culture. Cells werediluted to a density of 6×10⁵ cells/ml in 25 ml FreeStyle medium(Invitrogen, Carlsbad Calif.) 18-24 hours before transfection.Transfection complexes were prepared by adding 25 μg DNA to 1.25 ml PBS,adding 50 μl linear 25 kD polyethylenimine (PEI) (Polysciences,Warrington Pa.) dissolved in water at a concentration of 1 mg/ml, mixingthe solutions, and incubating the mixture for 1 hour at room temperaturebefore adding it to the cells to be transfected. Cells and theirsupernatants were harvested 3-6 days post-transfection and proteinexpression was evaluated by Western blot analysis.

Cell suspensions (1 ml) were pelleted then mixed with four parts XTsample buffer (Bio-Rad, Hercules Calif.). Following denaturation at 99°C. for 3 minutes, samples were either loaded onto a Criterion XTSDS-PAGE gel (Bio-Rad, Hercules Calif.) or stored at −20° C. Cellpellets were lysed by resuspension in 100 μl lysis buffer (1% NP-40; 50mM Tris-HCl, pH 8.0; 150 mM NaCl; and one tablet complete proteaseinhibitors (Roche, Indianapolis Ind.)). Lysed cells were pelleted bycentrifugation at 14,000 rpm and the proteins of the resulting clearedlysate, as well as the cell supernatants, were separated by SDS-PAGE andtransferred to a nitrocellulose membrane. Western blotting was performedby probing the membrane with a HRP-conjugated monoclonal antibodyspecific for the V5-epitope (Invitrogen, Carlsbad Calif.) or with apolyclonal rabbit-anti-MGD-CSF antibody (Five Prime Therapeutics, Inc.,South San Francisco Calif.). Bound rabbit-anti-MGD-CSF antibody wasdetected with polyclonal goat-anti-rabbit conjugated to horseradishperoxidase (Jackson Immuno Research, West Grove Pa.). Immunocomplexeswere visualized by incubating the membrane in chemiluminescencesubstrate (SuperSignal West Femto, Pierce, Rockford Ill.) and exposingit to light sensitive film.

As shown in FIGS. 4A and 4B, 293-6E cells expressed tagged MGD-CSFbetween 3 and 6 days after transfection. FIG. 4A shows the expression ofMGD-CSF tagged with V5H8 (CLN00732663) transiently transfected into293-6E cells. The left panel shows intracellular MGD-CSF. Expression wasmost prominent at day 3 post-transfection. The middle panel showsMGD-CSF secreted into the supernatant. Expression was most prominent atday 6 post-transfection. The right panel shows a quantitative positivecontrol (Positope, Invitrogen, Carlsbad, Calif.)

FIG. 4B shows the expression of MGD-CSF with a collagen secretorysequence and tagged with V5H8 (CLN00816424). The left panel showsintracellular MGD-CSF. Expression was observed at day 3 and continued toincrease through day 6 post-transfection. The middle panel shows MGD-CSFsecreted into the supernatant; its expression also increased from day 3to day 6. The right panel shows a quantitative positive control(Positope, Invitrogen, Carlsbad, Calif.).

In both FIGS. 4A and 4B, the protein loads of the cells and supernatantswere matched so that the gel loads of the left and middle panels reflectcomparable cell numbers. Thus, the amount of MGD-CSF shown in the middlepanels reflects the cells' secretory efficiency. Tagged protein detectedin the supernatant had a molecular weight of approximately 40 kD,whereas the intracellular protein had a molecular weight ofapproximately 37 kD, presumably due to incomplete glycosylation. Yieldsof the secreted protein differed depending on the construct design.CLN00732663 was expressed intracellularly at a low yield. The 37 kDform, and not the 40 kD form, was detected in cell culture supernatantsix days post transfection, also at a low yield (approximately 5-10ng/ml), possibly due to cell lysis. Replacing the endogenous signalpeptide with the exogenous secretion signal and an extended cleavableC-terminal tag increased expression and secretion at least 10-fold(CLN00816424). In addition, only the higher molecular weight proteinband was detectable in the supernatant of cell cultures transfected withCLN00816424, indicating the protein was secreted and did not originatefrom lysed cells.

Example 4 Proliferation and Viability of Transfected Cells

As shown in FIG. 5A and Table 1, 293-6E cells transiently transfectedwith CLN00816424 continued to proliferate from days 3 through 6post-transfection. The density of the cells in suspension culture wasmonitored by counting cells that excluded trypan blue using ahemocytometer from day 3 through day 6 following transfection withCLN00542945 (black), CLN00732663 (light grey), CLN00821867 (diagonalstripe), and CLN00816424 (cross-hatch), and compared to a control geneencoding secreted alkaline phosphatase (SEAP) (dark grey). Both thecontrol SEAP cells and the cells transfected with CLN00816424 (MGD-CSFwith a collagen leader) increased in number from day 3 through day 6.Cells transfected with CLN00542945 (untagged MGD-CSF), CLN00732663(V5H8-tagged MGD-CSF), or CLN00821867 (streptagged MGD-CSF) did notproliferate.

As shown in FIG. 5B, cells transiently transfected with CLN00816424(MGD-CSF with a collagen leader) remained viable. They maintainedgreater than 80% viability during 6 days in culture. The viability ofthe cells in suspension culture was monitored by Trypan Blue exclusionusing a hemocytometer from day 3 to day 6 following transfection withCLN00542945 (black), CLN00732663 (light grey), CLN00821867 (diagonalstripe), and CLN00816424 (cross-hatch), and compared to a control geneencoding secreted alkaline phosphatase (SEAP) (dark grey). Cellsexpressing MGD-CSF, CLN00732663, and CLN00821867, showed increased celltoxicity, evidenced by their decreased viability over time in culture.This toxicity is not specific to MGD-CSF cDNA in host 293 cells, butrather is observed only under certain culture conditions.

Example 5 Stable Transfection in Mammalian Cells

MGD-CSF was stably expressed by transfected adherent 293-T cells. Stabletransfection was performed in 293-T cells purchased from ATCC (ManassasVa.) and cultured in complete DMEM medium (DMEM medium supplemented with10% FBS (Mediatech, Herndon Va.); 100 U/ml penicillin, 100 μg/mlstreptomycin, and 2 mM glutamine (Invitrogen, Carlsbad Calif.)). The daybefore transfection, 1.25×10⁵ cells were seeded into a T-175 cultureflask (Corning, Acton MA) and incubated overnight at 37° C. with 5% CO₂.Cells were transfected by mixing 114 μl Fugene6 (Roche, Nutley N.J.)with 1.9 ml RPMI-1640 medium (Mediatech, Herndon Va.) and incubated for5 minutes at room temperature. Plasmid DNA (19 μg full length MGD-CSF inpIRESpuro3) (BD Biosciences, San Jose, Calif.) was added to theFugene/media mix and incubated for 15 minutes at room temperature. Thelipid/DNA mixture was transferred into the T-175 flask and incubatedwith the cells for 16 hours. The following day, the cells were detachedwith 0.25% trypsin (Invitrogen, Carlsbad Calif.) and expanded into threeT-175 flasks. After approximately 16 hours, the cells were attached tothe culture vessel and the selection reagent puromycin (Invivogen, SanDiego Calif.) was added to a final concentration of 10 mg/ml. Selectionmedium was changed once a week and the cell viability monitored for 4-6weeks. Expression was validated by Western blot analysis using thepolyclonal rabbit-anti-MGD-CSF antibody described above.

Adherent 293-T cells stably expressing MGD-CSF were adapted tosuspension culture in low-serum or serum free medium. Cells wereresuspended at a concentration of 10⁶/ml in FreeStyle medium(Invitrogen, Carlsbad Calif.) or in HyQ PF CHO LS medium (Hyclone, LoganUtah), respectively, and supplemented with 5% FBS (Mediatech, Herndon,Va.). Suspension cell cultures were maintained at a volume of 50 ml in a250 ml shake flask and cultured at 37° C. and 5% CO₂. The medium waschanged twice a week and the cells were maintained at a density at orbelow approximately 10⁶/ml. Cell viability was measured by Trypan Blueexclusion. When viability exceeded 80%, the serum concentration wasprogressively reduced to 3%, 2%, 1%, then serum free. Transfected cellsadapted to conditions of reduced or absent serum. Cell viability andprotein yields of 293-T cells transiently expressing untagged MGD-CSFwas considerably higher after four days in culture in HyQ-CHO media with1% FBS than the viability and yields observed after six days oftransient expression in 293-E cells cultured in Free Style media with 3%FBS.

Adherent 293-T cells maintained in FreeStyle medium with 3% FBS grewrelatively slowly; their viability was 67% after three passages. Incomparison, cells maintained in HyQ PF CHO LS medium with 1% FBS grewmore quickly; their viability was >85% after four passages. As shown inFIG. 6A, the protein yields of cultures in HyQ PF CHO LS medium and 1%FBS (right panel) were increased more than 10-fold compared to theyields from cells cultured in FreeStyle medium supplemented with 3% FBS(left panel), as determined by quantitative Western blot.

As shown in FIG. 6B, high protein yields were obtained from stablytransfected MGD-CSF 293-T cells adapted to grow in suspension culturewith low serum or with serum free media, as shown by Western blotanalysis. Stably transfected MGD-CSF 293-T cells adapted to grow insuspension culture with low serum secreted 8-fold more (1.3 μg ml)(panel 1). Those grown in serum free media secreted 4-fold more (650ng/ml) (panel 2) MGD-CSF into the culture supernatant within 6 days thanstably transfected MGD-CSF 293-T cells growing adherently as describedabove for 4 days (approximately 160 ng/ml) (panel 3). MGD-CSF expressedin E. coli and then purified served as a quantitative standard (panel4).

Example 6 Bioreactor Fermentation

As shown in FIG. 7, bioreactor fermentation improved MGD-CSFproductivity to approximately 10 μg/ml during the course of a 6-7 dayfermentation in a 10 liter bioreactor. Cells adapted to grow in lowserum were inoculated at a concentration of approximately 5×10⁵/ml into10 liters of HyQ PF CHO LS medium supplemented with 1% FBS. Fermentationwas monitored for 6 days and samples were prepared for gelelectrophoresis as described above. The left panel of FIG. 7 shows thepresence of MGD-CSF in a gel loaded with 22.5 μl sample per lane andstained with Coomassie Blue on days 1-6 post-inoculation. Bovine serumalbumin (BSA) is shown as a quantitative control in the right panel. Thearrow indicates the position of MGD-CSF.

Example 7 Protein Isolation

MGD-CSF was isolated from 293T cells grown in suspension cultures stablyexpressing the protein, as described above and shown in FIG. 8. Theculture supernatant was adjusted to 0.5 M NaCl, pH 5.0 (with HCl), thenconcentrated 5-fold with a cellulose membrane having a 10 kD molecularweight cut-off (Millipore, Billerica Mass.). The concentrate wasdialyzed against buffer A (10 mM acetic acid pH 5.0, 110 mM NaCl) andfractionated on a SP-Sepharose FF (Amersham, Piscataway N.J.) cationexchange column equilibrated with Buffer A. The protein was eluted witha linear gradient to 1.5 M NaCl and the fractions containing MGD-CSFwere dialyzed against buffer B (10 mM 1,3 diaminopropane pH 8.9, 30 mMNaCl).

This dialyzed SP-Pool was applied to a heparin Sepharose HP (Amersham,Piscataway, N.J.) column, equilibrated with buffer B and eluted with alinear gradient to 1.5 M NaCl. Fractions containing MGD-CSF weredialyzed against buffer C (10 mM bis-trispropane pH 7.4, 30 mM NaCl).This dialyzed Hep-Pool was fractionated on a Q-Sepharose FF (Amersham,Piscataway, N.J.) anion exchange column equilibrated in buffer C, andeluted with a linear gradient to 1.5 M NaCl. The fractions containingMGD-CSF were pooled and this Q-Pool was snap-frozen in liquid nitrogenand stored at −80C. This purification procedure recovered a yield of 12%of the expressed protein at >95% purity.

Example 8 Cysteine to Serine Mutational Analysis

The MGD-CSF sequence includes seven cysteine residues, located at aminoacid positions 35, 167, 176, 178, 179, 190, and 198. Based on acomparison of denaturing and nondenaturing gel electrophoresis resultsunder non-reducing conditions, MGD-CSF does not form disulfide-linkedoligomers. Therefore, at least one of the cysteine residues in thenative protein is expected to be unpaired. Unpaired cysteine residuesmay lead to improper protein folding and formation of covalentaggregates.

A set of seven muteins of the MGD-CSF protein was constructed,expressed, and characterized, in which each of the cysteines was mutatedto serine to understand its disulfide bond pattern. This analysis of thedisulfide bond pattern can determine whether eliminating one or morefree cysteine residues would produce an MGD-CSF protein with improvedproperties, for example, improved expression and secretion frommammalian cells, decreased aggregation of the purified protein, and/orthe potential to produce active recombinant MGD-CSF when expressed in E.coli.

DNA encoding each of the muteins was generated from a constructcomprising the collagen signal peptide and the nucleotide sequenceencoding mature MGD-CSF (CLN00848149). Protein was expressed in 293-Tcells, as described above. The supernatant was harvested and subjectedto reducing and nonreducing gel electrophoresis followed by Westernblotting with polyclonal antibody raised to the middle peptide epitopein human MGD-CSF.

As shown in FIG. 8B, the Western blot of the reducing gel was analyzedto determine relative expression levels of wild type human MGD-CSF (withnative or collagen signal peptide) and each of the Cys to Ser muteins.The secreted protein yield of wild type human MGD-CSF was observed to behigher with the collagen signal peptide than the native signal peptide.All of the muteins were observed to have at least a slightly decreasedyield of secreted protein as compared to wild type human MGD-CSF withthe collagen signal peptide. The yields of C179S and C190S weresignificantly decreased and C35S was not detectably expressed. Based onthese results, it is likely that C35, C179, and C190 participate indisulfide bonding in native MGD-CSF.

As further shown in FIG. 8B, the Western blot of the nonreducing gel wasanalyzed for changes in apparent molecular weight, as determined by therelative migration of the MGD-CSF species compared with proteinstandards. When a disulfide bond is disrupted, the apparent size of theprotein will typically increase under the denaturing conditions ofSDS-PAGE, and the magnitude of this increase will typically becorrelated with the distance between the two cysteine residues in theprimary sequence of the protein. The disruption of a disulfide bond maylead to the formation of higher molecular weight aggregated species.C167S is observed to have the same migration time as wild type MGD-CSF,while all of the other muteins have altered migration behavior. Thisindicates that C167 is likely the only unpaired cysteine in nativeMGD-CSF. C179S and C190S both primarily form higher molecular weightspecies. These muteins have the same changes in protein yield andmigration, suggesting that they are paired with one another in nativeMGD-CSF. C176S and C178S show the same slight decrease in migration,suggesting that they may be paired with each other in native MGD-CSF.Finally, C198S has a larger change in migration, suggesting that itspartner may be C35, which is located further away in the proteinsequence. The fact that C167 is likely the only unpaired cysteine innative MGD-CSF indicates it may be mutated to a serine or an alaninewith a resulting improvement in the yield of the expressed protein and adecrease in the heterogeneity of the recovered protein product.

Example 9 MGD-CSF Promotes Hematapoeitic Cell Proliferation

A. MGD-CSF Promotes NK Cell Proliferation

Mouse NK cells were purified from the spleens of C57BL6 10 week oldfemale mice using the NK cell isolation kit according to themanufacturer's instructions (Miltenyi Biotechnology Inc., AuburnCalif.). Approximately 30,000 purified NK cells were incubated withpurified MGD-CSF at concentrations from 0.01 to 10 μg/ml. NK cellnumbers were determined using the CellTiter-Glo Luminescent CellViability Assay Kit (Promega #G7571). As shown in FIG. 9, after fourdays of incubation in RPMI with 5% FBS, MGD-CSF specifically increasedthe proliferation and/or survival of NK cell numbers in a dose-dependentmanner.

Human NK cells were isolated and purified from blood enriched in buffycoat cells obtained from the Stanford Blood Center (Palo Alto, Calif.).The blood was diluted approximately 1:5 with PBS and Ficoll(Ficoll-Paque Plus, Amersham Biosciences; Piscataway, N.J.) added (12.5ml/tube) to multiple 50 ml conical tubes, each with 25 ml of dilutedblood. The Ficoll/blood mixture was centrifuged at 450×g for 30 minutes.The peripheral blood mononuclear cell (PBMC) layers were removed, washedwith DPBS 1× without calcium and magnesium (Mediatech, Inc., PrinceWilliam Co. VA) and pelleted at 1000 RPM for 10 minutes. The PBMCs werewashed three times in PBS by centrifugation at 1350 RPM for 10 minutesand resuspended in 40 ml PBS with 0.5% fetal calf serum (Gibco(Invitrogen), Carlsbad Calif.) and 2 mM EDTA (Sigma Aldrich, St. LouisMo.) (PBSFE).

NK cells were enriched from the PBMCs with a human NK Cell Isolation KitII (Miltenyi Biotechnology Inc., Auburn Calif.), as recommended by themanufacturer. This enrichment step utilized the “deplete” program of anautoMACST™ Separator (Miltenyi Biotechnology Inc., Auburn Calif.); thenegative fraction, representing enriched NK cells, was collected fromoutlet port “neg1.” These cells were centrifuged at 1350 RPM for 10minutes, the cell pellets resuspended in DMEM with 10% fetal calf serum,and diluted to a concentration of 1×10⁶ cells/ml. The cells wereincubated with the control and test agents described below for four daysat 37° C. in an atmosphere of 7% CO₂ in 96 well round bottom plates at acell concentration of 5×10⁴ cells in 50 μl DMEM with 10% fetal calfserum per well.

The effect of control and test agents on the proliferation of human NKcells prepared in this manner was determined in a screening assay bymeasuring the number of viable cells in the culture based onquantitation of ATP by measuring luciferase activity as described inPromega CellTitreGlo Technical Bulletin No. 288 (Promega, Madison Wis.).Quantitative results were read on an Lmax plate reader (MolecularDevices, Sunnyvale Calif.) at room temperature for 0.6 second/well. TheATP content of the wells was measured four days after plating.

As shown in FIG. 10, conditioned media from cells transfected withMGD-CSF and with cells transfected with plasmid DNA from cluster 190647,the source of the MGD-CSF clones, stimulated NK cell production.Interferon gamma (IFN-γ), interleukin 1 (IL-1), and GM-CSF were used asinternal positive controls. External positive controls, which includerecombinant interleukin 15, and external negative controls, whichinclude culture medium, are shown on the right and the left of FIG. 10.This screening assay identified MGD-CSF as an agent that stimulated theproduction of and/or stabilized the number of NK cells. This result wasseen in four independent repetitions of the screening assay. MGD-CSF didnot consistently induce the production of cytokines from NK cells. Itincreased or stabilized monocyte cell number but did not induce theproduction of cytokines from monocytic cells. MGD-CSF had no effect onthe number of activated T cells or B cells.

B. MGD-CSF Promotes Hematapoeitic Stem Cell Proliferation

MGD-CSF also stimulated the proliferation of bone marrow CD34⁺hematopoietic stem cells (HSC cells) (Cambrex, Inc., Baltimore Md.) inculture. As shown in FIG. 11, MGD-CSF increased proliferation in a dosedependent manner. HSC cells were grown in culture under stromal freeconditions at a density of 2.4×10⁴ cells per well in 12-well tissueculture dishes containing 1 ml/well RPMI (ATCC) supplemented with 5%heat inactivated fetal bovine serum (ATCC) and 10 ng/ml recombinanthuman stem cell factor (SCF), 10 ng/ml Flt3 ligand (Flt3L) (R&D Systems,Minneapolis Minn.) in a 5% CO₂ incubator at 37° C. for 1-2 weeks, washedwith PBS, lifted with 0.5 ml Versene (Gibco BRL, Gaithersburg Md.),washed again with PBS, resuspended in 1 ml PBS/0.1% BSA (Sigma, St.Louis Mo.) and counted with a hematocytometer. Purified MGD-CSFincreased their growth in a dose dependent manner from 20 ng/ml to 500ng/ml. MGD-CSF induced stem cell proliferation to a greater extent thanM-CSF and to a similar extent as G-CSF and GM-CSF.

C. MGD-CSF Promotes Myelocytic Cell Proliferation In Vitro

Human primary monocytes were purified from PBMC using a protocolmodified from a previously-described method (de Almeida, et al., Mem.Inst Oswaldo Cruz 95:221-223, 2000). To isolate human PBMC from blood,the buffy coat was diluted in a six-fold volume of PBS, then overlainonto 20-ml Ficoll in a 50 ml tube. The tubes were centrifuged at 2,000rpm at 22° C. for 20 minutes without the use of the centrifuge brake.The PMBC cells were collected from the interface, washed with PBS twicethen resuspended in RPMI 5% FBS and filtered through a BD Falcon cellstrainer. To purify the primary untouched monocytes from PBMC, six ml ofthe PBMC suspension (containing 70-120×10⁶ cells) was carefully andslowly overlain onto 10 ml hyperosmotic Percoll. The cells werecentrifuged at a speed of 580×g for 15 minutes without the use of thecentrifuge brake. Cells at the interface were collected and washed with50 ml of RPMI 5% FBS. This purified monocyte cell pellet was resuspendedin 50 ml RPMI 5% FBS.

The monocyte assay was performed by incubating approximately 30,000purified monocytes with MGD-CSF purified as described above. After fourdays of incubation in RPMI with 5% FBS, monocyte proliferation wasdetermined using the CellTiter-Glo Luminescent Cell Viability Assay Kit(Promega #G7571).

Conditioned medium from MGD-CSF transfected 293-T cells (MGD-CSF CM)promoted monocyte proliferation. The screening assay was performed induplicate plates in a 96-well plate format on monocytes activated bymouse IgG2a. Table 6 shows semiquantitative descriptions of the potencyof the activity of each clone to stimulate monocyte proliferation andthe degree of expression of each construct. MGD-CSF CM stimulatedmonocyte proliferation to approximately the same extent as GM-CSF.Results were considered significant when at least two standarddeviations from the median. The observed ED₅₀ was 3-5 ng/ml. MutantMGD-CSF proteins were also tested in this assay. The mutant cloneCLN00848185 demonstrated activity (potency) comparable to the wild typeprotein, and other mutant clones had slightly lower activities than thewild type protein, suggesting that some mutant proteins can be used astherapeutic proteins.

As shown in FIG. 12A, the stimulatory effect of MGD-CSF CM on monocyteproliferation was dose-dependent over a 10,000-fold range. The lowestdose of MGD-CSF CM tested, 0.01 μl, had no significant effect onmonocyte proliferation. Increasing the dose 10-fold to 0.1 μl MGD-CSF CMinduced cell proliferation to a significant level compared to controls.Further increasing the dose to 1 μl MGD-CSF CM and 10 μl MGD-CSF CMfurther increased monocyte proliferation in a dose-dependent manner. Nodose dependency was observed with the empty vector or the negativecontrols CLN003732 or FPT026. The effect of a single dose of 10 ng/mlGM-CSF, a stimulatory positive control, is shown, as well as the effectof the negative control IL-10, and of unconditioned medium.

As shown in FIG. 12B, both purified GM-CSF and conditioned media fromcells transfected with MGD-CSF stimulated human monycyte proliferation.Thus, MGD-CSF functions as an agonist of monocyte proliferation, inaddition to its role in the differentiation and growth of myeloid cellsand granulocytes. It may be used as a hematopoietic factor to enhancethe recovery of hematopoietic cells following chemotherapy or radiationtreatment and bone marrow transplantation in cancer patients.

D. MGD-CSF Promotes Myelocytic Cell Proliferation In Vivo

To understand the role of MGD-CSF in vivo, C57BL6 mice were injectedwith MGD-CSF plasmid DNA using a method described by Wang, et al. CancerRes. 63:9016-9022, 2003). The human cytochrome P450 3A4 promoter wasoperably linked to a nucleic acid molecule with the nucleotide sequenceof MGD-CSF and injected into the tail vein of a mouse in order totransfect the mouse's liver with MGD-CSF. The human 3A4 promoter wasused to drive the expression of MGD-CSF in mouse liver. A complete bloodcount (CBC) and differential analysis was performed on the control andexperimental groups in each of two independent experiments using aSerono Baker 9000 hematology analyzer.

The control group (Table 7A, animals 1-3) in the first experimentcomprised three uninjected mice age matched to the experimental mice.The experimental group (Table 7A, animals 4-6) in the first experimentcomprised three mice injected with naked MGD-CSF DNA via the tail vein.Blood samples were collected on day 14 following injection. As shown inTable 7A, the injected mice had elevated monocyte counts compared to thecontrols. Control animals 1, 2, and 3 had 94, 84, and 52 monocytes/μlblood, respectively. Experimental animals 4 and 6 had 216 and 268monocytes/μl blood, respectively. No meaningful monocyte count wasobtained for animal 3.

The control group (Table 7B, animals 1-6) in the second experimentcomprised six mice age matched to the experimental mice and injected viathe tail vein with a LacZ construct. The experimental group (Table 7B,animals 7-12) in the second experiment comprised six mice injected withnaked MGD-CSF DNA via the tail vein. Blood samples were collected on day21 following injection. As shown in Table 7B, the injected mice hadelevated monocyte counts compared to the controls. None of the controlanimals had detectable monocyte levels. Four of the six experimentalanimals had detectable monocyte levels, ranging from 50-78 monocytes/μl.These results demonstrate that MGD-CSF increased myeloid cell numbers invivo.

Example 10 FACS Analysis of the Effect of MGD-CSF on HematopoieticDifferentiation

In vitro granulocyte, monocyte, and dendritic cell development assaysfurther revealed the function of MGD-CSF in hematopoeisis. Human bonemarrow CD34⁺ hematopoietic stem cells (HSC cells) (Cambrex, Inc.,Baltimore Md.) were cultured as described above.

Differentiation was determined by fluorescence activated cell sorting(FACS) analysis using fluorescently labeled antibodies to detect thedifferentiation markers on the granulocyte cell surface (Kavathas etal., Proc. Natl. Acad. Sci. 80:524-528 (1983)). After one week culturein either the presence or absence of MGD-CSF, G-CSF, GM-CSF, or M-CSF,the BM CD34⁺ cells were washed once with PBS, lifted with 0.5 ml Versene(Gibco BRL, Gaithersburg Md.), washed with 1 ml PBS/0.1% BSA (Sigma, St.Louis Mo.), resuspended in 0.2 ml PBS/0.1% BSA (Sigma, St. Louis Mo.),and aliquoted (50 μl per well) into a 96-well plate for FACS staining.Cells were incubated with fluorescent-conjugated antibodies for 15minutes at 4° C. After washing twice with 150 μl PBS/0.1% BSA, the cellswere analyzed with a FACS Calibur, per manufacturer's instruction(Becton Dickinson, Franklin Lakes N.J.). 10 ng/ml G-CSF, 10 ng/ml M-CSF,or 30 ng/ml GM-CSF (from R&D Systems, Minneapolis Minn.) served forpositive controls of the effects of known growth factors.Fluorescent-conjugated antibodies specific for granulocyte, monocyte, ordendritic lineage-specific surface markers were purchased from BDBiosciences, (San Jose, Calif.) and used to determine the effect ofMGD-CSF on differentiation of HSC cells to granulocytic, monocytic, anddendritic lineages.

A. Granulocyte Differentiation

As shown in FIG. 13, MGD-CSF stimulated the differentiation ofgranulocytes from undifferentiated cells to differentiated granulocytespossessing the differentiation markers CD67⁺ and CD24⁺. As a negativecontrol, the baseline level of granulocyte differentiation in thepresence of empty vector and the absence of cytokine was measured to be1.2%. The positive control, G-CSF, stimulated 55% of the granulocytes todifferentiate. MGD-CSF CM stimulated 41% of the granulocytes todifferentiate (arrow). The effect of MGD-CSF was synergistic with thatof G-CSF, in the presence of both, 64% of the granulocytes werestimulated to differentiate.

CD24 and CD15 antibodies were used to monitor granulocytedifferentiation. The CD24 antibody reacted with a 35-45 kDa two-chainglycoprotein expressed on the surface of B cells and granulocytes. TheCD15 antibody reacted with 3-fucosyl-N-acetyllactosamine (3-FAL), a 220kDa carbohydrate structure, also known as X-hapten. 3-FAL was expressedon 95% of the granulocytes examined, including neutrophils andeosinophils, and to a varying degree on monocytes, but not onlymphocytes or basophils. CD15 plays a role in mediating phagocytosis,bactericidal activity and chemotaxis. Cells positive for both CD24 andCD15 represent granulocytes which have differentiated from the BM CD34⁺hematopoietic progenitor cells.

As shown in FIG. 14, 20 ng/ml and 100 ng/ml MGD-CSF induced 3%differentiation to CD15⁺/CD24⁺ granulocytes, and 500 ng/ml MGD-CSFinduced 8% differentiation to CD15⁺/CD24⁺ granulocytes. The positivecontrols G-CSF and GM-CSF both induced 12% of bone marrow CD34⁺ cells todifferentiate into CD15/CD24 positive granulocytes.

B. Monocyte Differentiation

As shown in FIG. 15, MGD-CSF stimulated the differentiation of monocytesfrom undifferentiated cells to differentiated monocytes possessing theCD14⁺ marker. As a negative control, the baseline level of monocytedifferentiation in the presence of empty vector and the absence ofcytokine was measured to be 24%. GM-CSF stimulated 20% of the monocytesto differentiate. MGD-CSF stimulated 45% of the monocytes todifferentiate (arrow). Although GM-CSF alone had no effect on thesecells, when combined with MGD-CSF, its effect was synergistic; in thepresence of both, 55% of the monocytes were stimulated to differentiate.

As shown in FIG. 16, CD14 and CD16 antibodies were used to monitormonocyte differentiation. The CD14 antibody reacted with a 53-55 kDglycosylphosphatidylinositol (GPI)-anchored single chain glycoproteinexpressed at high levels on monocytes. Additionally, the CD14 antibodyreacted with some macrophages. The CD16 antibody reached with the 50-65kDa transmembrane form of IgG Fc receptor (FcgRIII). CD16 antigen wasexpressed on monocytes, macrophages, granulocytes, and NK cells.Monocytes can be divided into two subsets according to their CD16expression; resident monocytes are CD14⁺CD16⁻ and inflammatory monocytesare CD14^(low)CD16⁺. 20 ng/ml MGD-CSF induced 30% differentiation, 100ng/ml MGD-CSF induced 30% differentiation, and 500 ng/ml induced 53%differentiation to either CD14⁺CD16⁻ or CF14^(low)CD16⁺ monocytes. Thepositive controls G-CSF, GM-CSF, and M-CSF promoted 41%, 38%, and 51%CD14 differentiation, respectively.

C. Dendritic Cell Differentiation

As shown in FIG. 17, MGD-CSF stimulated the differentiation of dendriticcells from undifferentiated cells to differentiated dendritic cellspossessing the CD86 and CD1 markers. As a negative control, the baselinelevel of dendritic cell differentiation in the presence of empty vectorwas measured to be 4%. MGD-CSF stimulated 22% of the undifferentiatedcells to differentiate into dendritic cells.

Example 11 MGD-CSF Promotes Bone Marrow Colony Formation

To assess stimulatory effects of MGD-CSF on human bone marrow derivedmyeloid (CFU-G, CFU-M, and CFU-GM) progenitor proliferation, colonyformation assays were performed. The positive controls for stimulationof myeloid progenitors were the addition of G-CSF at 0.1 ng/ml, 1 ng/mland 10 ng/ml and GM-CSF at 0.01 ng/ml, 0.1 ng/ml and 3 ng/ml. MGD-CSFprotein was diluted into methylcellulose-based media for each testconcentration to final concentrations of 20, 100, and 500 ng/ml. Cellswere added such that each of three replicate cultures contained 3×10⁴cells. The replicate cultures were incubated at 37° C., 5% CO₂ for 14days, then counted, photographed, classified on the basis of morphologyas CFU-G, CFU-M, or CFU-GM, and FACS analysis was performed. Statisticalanalyses were performed to assess changes in colony number, size, andmorphology. MGD-CSF promoted formation of CFU-G, CFU-M, CFU-GM, andtotal colony formation. In addition, as further described below, MGD-CSFpromoted large CFU-M colonies distinct from those promoted by G-CSF orGM-CSF. These results suggest that MGD-CSF enhanced early hematopoieticprogenitor formation.

FIG. 18A and FIG. 18B show the effect of MGD-CSF on human bone marrowcolony formation in the absence of exogenous cytokines. As shown in FIG.18A, purified MGD-CSF had little effect on granulocyte colony formation(CFU-G) compared to G-CSF and GM-CSF. MGD-CSF stimulated monocyte colonyformation (CFU-M) in a dose-dependent manner. As shown in FIG. 18B,MGD-CSF stimulated granulocyte-monocyte colony formation CFU-GM in adose-dependent manner. MGD-CSF also stimulated total colony formingcapacity (CFC) in a dose-dependent manner. FIG. 18C shows the effect ofMGD-CSF on human bone marrow colony formation in the presence of thecytokines IL-3 and stem cell factor (SCF). Under those conditions,purified MGD-CSF stimulated CFU-G, CFU-GM, CFU-M, and total CFC in adose-dependent manner. The distribution of myeloid progenitors in thepresence of MGD-CSF was distinct from that of G-CSF or GM-CSF.

Example 12 Profile of Biological Activities of MGD-CSF

As shown in FIG. 19, MGD-CSF was tested for its biological effects inassays that measure non-activated B cell proliferation (BPro4), abilityto stimulate glucose uptake by adipocytes (Gu2Gy3T3), unactivatedmonocyte proliferation (MonPro4), NK cell proliferation and/or survival(NKGlo), T cell proliferation (TPro4), activated primary B cellproliferation (aBPro4), activated primary monocyte proliferation(aMonPro3), and activated primary T cell proliferation (aTPro4). Resultswere considered significant if they were at least two standarddeviations from the median. MGD-CSF specifically stimulated theproliferation of NK cells and unactivated monocytes, without stimulatingthe proliferation of activated monocytes or the proliferation of eitheractivated or unactivated B cells or T cells.

Example 13 Profile of MGD-CSF-Induced Cytokine Secretion

As shown in FIG. 20, MGD-CSF was tested for its biological effect oncytokine secretion from NK cells. Assays were performed as described inExample 11. Conditioned medium was removed at the end of the experimentto determine the type and amount of cytokine secretion, using theLuminex cytokine assay kit from Linco, Inc. (St. Charles, Mo.) accordingto the manufacturer's instructions. Results were considered significantif they were at least two standard deviations from the median. MGD-CSFstimulated the secretion of GM-CSF, IL-12, and IL-13.

Example 14 MGD-CSF Stimulated CFU-M Differentiation from HSC Cells

MGD-CSF increased the size as well as the number of myeloid coloniesformed from human bone marrow cells in a dose-dependent manner. The topleft panel of FIG. 21 shows a representative photograph of CFU-Mcolonies observed in bone marrow cells cultured the absence of cytokine(buffer). Evidence of colony formation is weak or absent. The top middlepanel shows a representative photograph of CFU-M colonies induced byGM-CSF; colony formation was apparent. The top right panel shows arepresentative photograph which demonstrates that G-CSF does notstimulate CFU-M formation. The bottom three panels of FIG. 16 showrepresentative photographs of CFU-Ms induced by MGD-CSF. Both the numberand the size increased in a dose-dependent manner between 20 ng/ml and500 ng/ml MGD-CSF. The cells were examined and photographed with anAxiovert 25 microscope and AxioCam HRc (both from Carl Zeiss, Gottingen,Germany) using a 40× lens. Cells were visualized with a Zeiss KS300 3.0digital imaging system.

The size of the colonies induced by MGD-CSF were larger than thoseinduced by GM-CSF. Approximately 10% of the colonies induced by MGD-CSFwere extremely large, in the range of 100-2000 microns. MGD-CSF inducedthese large colonies both in the presence and absence of the cytokinesSCF and IL-3. These data show that MGD-CSF promoted the formation ofearly myeloid progenitors, including progenitors earlier than GM-CSF orG-CSF. They also suggest that MGD-CSF promotes the differentiation ofeither or both of the M1 or M2 macrophage lineage.

Example 15 MGD-CSF Stimulated HSC Differentiation to Dendritic Cells

Human bone marrow CD34⁺ cells (Cambrex, Inc., Baltimore Md.) were platedon 24-well cell culture plates in serum-free X-vivo 20 medium (Cambrex,Inc., Baltimore Md.), and treated with vector control conditioned medium(CM) or MGD-CSF CM. The cells were examined and photographed with anAxiovert 25 microscope and AxioCam HRc (both from Carl Zeiss, Gottingen,Germany) using a 40× lens. Cells were visualized with a Zeiss KS300 3.0digital imaging system.

As shown in FIG. 22, increasing numbers of elongated cells were observedin the MGD-CSF-treated cultures with increasing MGD-CSF concentrations.Anti-CD1a antibody was used to determine differentiation into dendriticcells. Treating the cultures with MGD-CSF for two weeks induced thedifferentiation of 20% of the human bone marrow CD34⁺ into CD1a positivedendritic cells. MGD-CSF induced the differentiation of dendritic cellsfrom human bone marrow cells in a dose-dependent manner (FIG. 22). Thecells shown in all four panels were examined and photographed with anAxiovert 25 microscope and AxioCam HRc (both from Carl Zeiss, Gottingen,Germany) using a 40× lens. Cells were visualized with a Zeiss KS300 3.0digital imaging system.

The top left panel of FIG. 22 shows a representative photograph of bonemarrow cells cultured the absence of cytokine (medium). The cells aretypically small and rounded. The top right panel shows a representativephotograph of the bone marrow cells elongating and flattening to moreclosely resemble dendritic cells, in response to 20 ng/ml MGD-CSF. Thebottom left panel shows a representative photograph which demonstratesthat a higher dose of MGD-CSF, 100 ng/ml, has a more pronounced effecton the morphology of the HSC cells. The cells are larger and moreelongated. The bottom right panel shows that a concentration of 500ng/ml MGD-CSF resulted in large, flat, elongated cells with themorphological appearance of dendritic cells.

Example 16 MGD-CSF Gene Expression

The differential level of gene expression was compared in individualhuman cancer tissue specimens by interrogating a proprietary oncologydatabase from GeneLogic, using the Affymetrix GeneChip® array platform,the Human Genome U133 and U133Plus_(—)2 (Affymetrix, Inc, Santa Clara,Calif.) with probe 237046_x_at. It was also compared by interrogatingmicroarray chips designed by Five Prime Therapeutics, Inc. with probesPRB107386 and PRB107386_at. These probes were used to determine theexpression of MGD-CSF in the tissues of patients with hyperproliferativehematologic abnormalities. This analysis identified differential geneexpression patterns between different tissue types and different diseasestages. Table 8, column 3 lists the number of disease specimens thattested positive for the presence of MGC34647 (MGC34647 Positive). Table8, column 4 lists the number of specimens examined (Total Gene Logic).MGD-CSF was expressed in most patients with myelodysplastic syndrome.The percent of patients expressing MGD-CSF varied with the observedpathology and was highest in patients with refractory anemia with excessblasts or ringed sideroblasts. Half of the patients with acute B-celllymphoblastic leukemia expressed MGD-CSF. A subset of patients withacute myeloid leukemia expressed MGD-CSF. The percentage varied from14-25%, depending on the pathological presentation of the disease.MGD-CSF was generally not expressed in patients with chronic myeloidleukemia, chronic lymphocytic leukemia, or acute promyelocytic leukemia.

SEQUENCE LISTING

A sequence listing transmittal sheet and a sequence listing in paperformat accompanies this application.

Tables

TABLE 1 SEQ. ID. NOS.: 1-271 SEQ. ID. SEQ. ID SEQ. ID. FP ID NO.: (N1)NO.: (P1) NO.: (N0) Source ID Type HG1015544 SEQ. ID. SEQ. ID.CLN00542945 NO.: 1 NO.: 7 HG1015545 SEQ. ID. SEQ. ID. CLN00542945_exon4NO.: 2 NO.: 8 HG1015596 SEQ. ID. SEQ. ID. CLN00542945_mature NO.: 3 NO.:9 peptide HG1015546 SEQ. ID. SEQ. ID. SEQ. ID. NP_689669 NO.: 4 NO.: 10NO.: 13 HG1015597 SEQ. ID. SEQ. ID. CLN00542945_fragment NO.: 5 NO.: 11HG1019016 SEQ. ID. SEQ. ID. NP_689669_fragment NO.: 6 NO.: 12 HG1018265SEQ. ID. collagen_leader_seq leader sequence NO.: 14 HG1018268 SEQ. ID.112907: HMM_SP leader sequence NO.: 15 21594845_1-17 HG1018269 SEQ. ID.112907: leader sequence NO.: 16 21594845_1-13 HG1018270 SEQ. ID. 112907:leader sequence NO.: 17 21594845_1-19 HG1018271 SEQ. ID. 112907: leadersequence NO.: 18 21594845_1-16 HG1018272 SEQ. ID. 112907: leadersequence NO.: 19 21594845_1-15 HG1018274 SEQ. ID. 13325208: HMM_SPleader sequence NO.: 20 13325207_1-30 HG1018275 SEQ. ID. 13325208:leader sequence NO.: 21 13325207_1-25 HG1018276 SEQ. ID. 13325208:leader sequence NO.: 22 13325207_1-33 HG1018277 SEQ. ID. 13325208:leader sequence NO.: 23 13325207_1-24 HG1018278 SEQ. ID. 13325208:leader sequence NO.: 24 13325207_1-26 HG1018279 SEQ. ID. 13325208:leader sequence NO.: 25 13325207_1-32 HG1018280 SEQ. ID. 13325208:leader sequence NO.: 26 13325207_1-27 HG1018281 SEQ. ID. 13325208:leader sequence NO.: 27 13325207_1-23 HG1018282 SEQ. ID. 13325208:leader sequence NO.: 28 13325207_1-35 HG1018284 SEQ. ID. 13938307:HMM_SP leader sequence NO.: 29 13938306_1-24 HG1018285 SEQ. ID.13938307: leader sequence NO.: 30 13938306_1-21 HG1018287 SEQ. ID.14718453: HMM_SP leader sequence NO.: 31 14718452_1-19 HG1018288 SEQ.ID. 14718453: leader sequence NO.: 32 14718452_1-15 HG1018289 SEQ. ID.14718453: leader sequence NO.: 33 14718452_1-17 HG1018291 SEQ. ID.15929966: HMM_SP leader sequence NO.: 34 15929965_1-23 HG1018293 SEQ.ID. 16356651: leader sequence NO.: 35 16356650_1-21 HG1018294 SEQ. ID.16356651: leader sequence NO.: 36 16356650_1-17 HG1018296 SEQ. ID.18204192: HMM_SP leader sequence NO.: 37 18204191_1-19 HG1018297 SEQ.ID. 18204192: leader sequence NO.: 38 18204191_1-22 HG1018298 SEQ. ID.18204192: leader sequence NO.: 39 18204191_1-18 HG1018299 SEQ. ID.18204192: leader sequence NO.: 40 18204191_1-16 HG1018300 SEQ. ID.18204192: leader sequence NO.: 41 18204191_1-14 HG1018302 SEQ. ID.23503038: leader sequence NO.: 42 15778555_1-20 HG1018303 SEQ. ID.23503038: leader sequence NO.: 43 15778555_1-16 HG1018304 SEQ. ID.23503038: leader sequence NO.: 44 15778555_1-21 HG1018306 SEQ. ID.27479535: HMM_SP leader sequence NO.: 45 27479534_1-24 HG1018307 SEQ.ID. 27479535: leader sequence NO.: 46 27479534_1-20 HG1018308 SEQ. ID.27479535: leader sequence NO.: 47 27479534_1-26 HG1018309 SEQ. ID.27479535: leader sequence NO.: 48 27479534_1-21 HG1018310 SEQ. ID.27479535: leader sequence NO.: 49 27479534_1-23 HG1018312 SEQ. ID.37182960: HMM_SP leader sequence NO.: 50 37182959_1-24 HG1018313 SEQ.ID. 37182960: leader sequence NO.: 51 37182959_1-19 HG1018314 SEQ. ID.37182960: leader sequence NO.: 52 37182959_1-22 HG1018315 SEQ. ID.37182960: leader sequence NO.: 53 37182959_1-20 HG1018316 SEQ. ID.37182960: leader sequence NO.: 54 37182959_1-26 HG1018317 SEQ. ID.37182960: leader sequence NO.: 55 37182959_1-21 HG1018319 SEQ. ID.7437388: HMM_SP leader sequence NO.: 56 1208426_1-24 HG1018320 SEQ. ID.7437388: leader sequence NO.: 57 1208426_1-23 HG1018322 SEQ. ID.NP_000286: HMM_SP leader sequence NO.: 58 NM_000295_1-24 HG1018323 SEQ.ID. NP_000286: leader sequence NO.: 59 NM_000295_1-18 HG1018324 SEQ. ID.NP_000286: leader sequence NO.: 60 NM_000295_1-23 HG1018325 SEQ. ID.NP_000286: leader sequence NO.: 61 NM_000295_1-17 HG1018327 SEQ. ID.NP_000396: HMM_SP leader sequence NO.: 62 NM_000405_1-23 HG1018328 SEQ.ID. NP_000396: leader sequence NO.: 63 NM_000405_1-18 HG1018329 SEQ. ID.NP_000396: leader sequence NO.: 64 NM_000405_1-25 HG1018330 SEQ. ID.NP_000396: leader sequence NO.: 65 NM_000405_1-20 HG1018331 SEQ. ID.NP_000396: leader sequence NO.: 66 NM_000405_1-21 HG1018333 SEQ. ID.NP_000495: HMM_SP leader sequence NO.: 67 NM_000504_1-23 HG1018334 SEQ.ID. NP_000495: leader sequence NO.: 68 NM_000504_1-19 HG1018335 SEQ. ID.NP_000495: leader sequence NO.: 69 NM_000504_1-20 HG1018336 SEQ. ID.NP_000495: leader sequence NO.: 70 NM_000504_1-15 HG1018337 SEQ. ID.NP_000495: leader sequence NO.: 71 NM_000504_1-21 HG1018338 SEQ. ID.NP_000495: leader sequence NO.: 72 NM_000504_1-17 HG1018340 SEQ. ID.NP_000573: HMM_SP leader sequence NO.: 73 NM_000582_1-18 HG1018341 SEQ.ID. NP_000573: leader sequence NO.: 74 NM_000582_1-16 HG1018342 SEQ. ID.NP_000573: leader sequence NO.: 75 NM_000582_1-15 HG1018344 SEQ. ID.NP_000574: HMM_SP leader sequence NO.: 76 NM_000583_1-16 HG1018345 SEQ.ID. NP_000574: leader sequence NO.: 77 NM_000583_1-14 HG1018347 SEQ. ID.NP_000591: HMM_SP leader sequence NO.: 78 NM_000600_1-25 HG1018348 SEQ.ID. NP_000591: leader sequence NO.: 79 NM_000600_1-24 HG1018349 SEQ. ID.NP_000591: leader sequence NO.: 80 NM_000600_1-27 HG1018351 SEQ. ID.NP_000598: HMM_SP leader sequence NO.: 81 NM_000607_1-18 HG1018353 SEQ.ID. NP_000604: leader sequence NO.: 82 NM_000613_1-19 HG1018354 SEQ. ID.NP_000604: leader sequence NO.: 83 NM_000613_1-25 HG1018355 SEQ. ID.NP_000604: leader sequence NO.: 84 NM_000613_1-21 HG1018356 SEQ. ID.NP_000604: leader sequence NO.: 85 NM_000613_1-23 HG1018357 SEQ. ID.NP_000604: leader sequence NO.: 86 NM_000613_1-31 HG1018359 SEQ. ID.NP_000726: HMM_SP leader sequence NO.: 87 NM_000735_1-26 HG1018360 SEQ.ID. NP_000726: leader sequence NO.: 88 NM_000735_1-24 HG1018362 SEQ. ID.NP_000884: HMM_SP leader sequence NO.: 89 NM_000893_1-18 HG1018363 SEQ.ID. NP_000884: leader sequence NO.: 90 NM_000893_1-19 HG1018364 SEQ. ID.NP_000884: leader sequence NO.: 91 NM_000893_1-16 HG1018365 SEQ. ID.NP_000884: leader sequence NO.: 92 NM_000893_1-23 HG1018367 SEQ. ID.NP_000909: HMM_SP leader sequence NO.: 93 NM_000918_1-17 HG1018369 SEQ.ID. NP_000930: HMM_SP leader sequence NO.: 94 NM_000939_1-23 HG1018370SEQ. ID. NP_000930: leader sequence NO.: 95 NM_000939_1-26 HG1018372SEQ. ID. NP_000945: HMM_SP leader sequence NO.: 96 NM_000954_1-23HG1018373 SEQ. ID. NP_000945: leader sequence NO.: 97 NM_000954_1-22HG1018374 SEQ. ID. NP_000945: leader sequence NO.: 98 NM_000954_1-18HG1018376 SEQ. ID. NP_001176: leader sequence NO.: 99 NM_001185_1-18HG1018377 SEQ. ID. NP_001176: leader sequence NO.: 100 NM_001185_1-20HG1018378 SEQ. ID. NP_001176: leader sequence NO.: 101 NM_001185_1-21HG1018379 SEQ. ID. NP_001176: leader sequence NO.: 102 NM_001185_1-17HG1018381 SEQ. ID. NP_001266: HMM_SP leader sequence NO.: 103NM_001275_1-18 HG1018382 SEQ. ID. NP_001266: leader sequence NO.: 104NM_001275_1-15 HG1018383 SEQ. ID. NP_001266: leader sequence NO.: 105NM_001275_1-14 HG1018385 SEQ. ID. NP_001314: HMM_SP leader sequence NO.:106 NM_001323_1-26 HG1018386 SEQ. ID. NP_001314: leader sequence NO.:107 NM_001323_1-18 HG1018387 SEQ. ID. NP_001314: leader sequence NO.:108 NM_001323_1-20 HG1018388 SEQ. ID. NP_001314: leader sequence NO.:109 NM_001323_1-28 HG1018389 SEQ. ID. NP_001314: leader sequence NO.:110 NM_001323_1-21 HG1018390 SEQ. ID. NP_001314: leader sequence NO.:111 NM_001323_1-23 HG1018392 SEQ. ID. NP_001822: leader sequence NO.:112 NM_001831_1-22 HG1018393 SEQ. ID. NP_001822: leader sequence NO.:113 NM_001831_1-18 HG1018394 SEQ. ID. NP_001822: leader sequence NO.:114 NM_001831_1-14 HG1018396 SEQ. ID. NP_002206: leader sequence NO.:115 NM_002215_1-24 HG1018397 SEQ. ID. NP_002206: leader sequence NO.:116 NM_002215_1-29 HG1018398 SEQ. ID. NP_002206: leader sequence NO.:117 NM_002215_1-30 HG1018399 SEQ. ID. NP_002206: leader sequence NO.:118 NM_002215_1-23 HG1018400 SEQ. ID. NP_002206: leader sequence NO.:119 NM_002215_1-31 HG1018402 SEQ. ID. NP_002300: HMM_SP leader sequenceNO.: 120 NM_002309_1-22 HG1018403 SEQ. ID. NP_002300: leader sequenceNO.: 121 NM_002309_1-23 HG1018405 SEQ. ID. NP_002336: HMM_SP leadersequence NO.: 122 NM_002345_1-18 HG1018406 SEQ. ID. NP_002336: leadersequence NO.: 123 NM_002345_1-15 HG1018407 SEQ. ID. NP_002336: leadersequence NO.: 124 NM_002345_1-17 HG1018408 SEQ. ID. NP_002336: leadersequence NO.: 125 NM_002345_1-14 HG1018410 SEQ. ID. NP_002402: HMM_SPleader sequence NO.: 126 NM_002411_1-18 HG1018412 SEQ. ID. NP_002505:HMM_SP leader sequence NO.: 127 NM_002514_1-30 HG1018413 SEQ. ID.NP_002505: leader sequence NO.: 128 NM_002514_1-32 HG1018414 SEQ. ID.NP_002505: leader sequence NO.: 129 NM_002514_1-28 HG1018415 SEQ. ID.NP_002505: leader sequence NO.: 130 NM_002514_1-27 HG1018416 SEQ. ID.NP_002505: leader sequence NO.: 131 NM_002514_1-31 HG1018418 SEQ. ID.NP_002892: HMM_SP leader sequence NO.: 132 NM_002901_1-26 HG1018419 SEQ.ID. NP_002892: leader sequence NO.: 133 NM_002901_1-22 HG1018420 SEQ.ID. NP_002892: leader sequence NO.: 134 NM_002901_1-29 HG1018421 SEQ.ID. NP_002892: leader sequence NO.: 135 NM_002901_1-24 HG1018422 SEQ.ID. NP_002892: leader sequence NO.: 136 NM_002901_1-23 HG1018424 SEQ.ID. NP_002893: HMM_SP leader sequence NO.: 137 NM_002902_1-25 HG1018425SEQ. ID. NP_002893: leader sequence NO.: 138 NM_002902_1-19 HG1018426SEQ. ID. NP_002893: leader sequence NO.: 139 NM_002902_1-22 HG1018427SEQ. ID. NP_002893: leader sequence NO.: 140 NM_002902_1-18 HG1018428SEQ. ID. NP_002893: leader sequence NO.: 141 NM_002902_1-20 HG1018429SEQ. ID. NP_002893: leader sequence NO.: 142 NM_002902_1-21 HG1018430SEQ. ID. NP_002893: leader sequence NO.: 143 NM_002902_1-23 HG1018432SEQ. ID. NP_005133: HMM_SP leader sequence NO.: 144 NM_005142_1-19HG1018433 SEQ. ID. NP_005133: leader sequence NO.: 145 NM_005142_1-18HG1018434 SEQ. ID. NP_005133: leader sequence NO.: 146 NM_005142_1-20HG1018435 SEQ. ID. NP_005133: leader sequence NO.: 147 NM_005142_1-24HG1018436 SEQ. ID. NP_005133: leader sequence NO.: 148 NM_005142_1-16HG1018437 SEQ. ID. NP_005133: leader sequence NO.: 149 NM_005142_1-17HG1018438 SEQ. ID. NP_005133: leader sequence NO.: 150 NM_005142_1-14HG1018440 SEQ. ID. NP_005445: HMM_SP leader sequence NO.: 151NM_005454_1-17 HG1018442 SEQ. ID. NP_005555: HMM_SP leader sequence NO.:152 NM_005564_1-18 HG1018443 SEQ. ID. NP_005555: leader sequence NO.:153 NM_005564_1-20 HG1018444 SEQ. ID. NP_005555: leader sequence NO.:154 NM_005564_1-15 HG1018446 SEQ. ID. NP_005690: HMM_SP leader sequenceNO.: 155 NM_005699_1-29 HG1018447 SEQ. ID. NP_005690: leader sequenceNO.: 156 NM_005699_1-24 HG1018448 SEQ. ID. NP_005690: leader sequenceNO.: 157 NM_005699_1-28 HG1018450 SEQ. ID. NP_006560: HMM_SP leadersequence NO.: 158 NM_006569_1-19 HG1018451 SEQ. ID. NP_006560: leadersequence NO.: 159 NM_006569_1-18 HG1018452 SEQ. ID. NP_006560: leadersequence NO.: 160 NM_006569_1-21 HG1018454 SEQ. ID. NP_006856: HMM_SPleader sequence NO.: 161 NM_006865_1-15 HG1018456 SEQ. ID. NP_036577:HMM_SP leader sequence NO.: 162 NM_012445_1-26 HG1018457 SEQ. ID.NP_036577: leader sequence NO.: 163 NM_012445_1-25 HG1018458 SEQ. ID.NP_036577: leader sequence NO.: 164 NM_012445_1-24 HG1018459 SEQ. ID.NP_036577: leader sequence NO.: 165 NM_012445_1-28 HG1018461 SEQ. ID.NP_055070: HMM_SP leader sequence NO.: 166 NM_014255_1-20 HG1018462 SEQ.ID. NP_055070: leader sequence NO.: 167 NM_014255_1-18 HG1018463 SEQ.ID. NP_055070: leader sequence NO.: 168 NM_014255_1-16 HG1018465 SEQ.ID. NP_055582: HMM_SP leader sequence NO.: 169 NM_014767_1-24 HG1018466SEQ. ID. NP_055582: leader sequence NO.: 170 NM_014767_1-19 HG1018467SEQ. ID. NP_055582: leader sequence NO.: 171 NM_014767_1-22 HG1018468SEQ. ID. NP_055582: leader sequence NO.: 172 NM_014767_1-20 HG1018469SEQ. ID. NP_055582: leader sequence NO.: 173 NM_014767_1-26 HG1018470SEQ. ID. NP_055582: leader sequence NO.: 174 NM_014767_1-21 HG1018472SEQ. ID. NP_055697: HMM_SP leader sequence NO.: 175 NM_014882_1-18HG1018474 SEQ. ID. NP_056965: HMM_SP leader sequence NO.: 176NM_015881_1-18 HG1018475 SEQ. ID. NP_056965: leader sequence NO.: 177NM_015881_1-19 HG1018476 SEQ. ID. NP_056965: leader sequence NO.: 178NM_015881_1-22 HG1018477 SEQ. ID. NP_056965: leader sequence NO.: 179NM_015881_1-16 HG1018478 SEQ. ID. NP_056965: leader sequence NO.: 180NM_015881_1-21 HG1018480 SEQ. ID. NP_057603: leader sequence NO.: 181NM_016519_1-26 HG1018481 SEQ. ID. NP_057603: leader sequence NO.: 182NM_016519_1-28 HG1018483 SEQ. ID. NP_149439: HMM_SP leader sequence NO.:183 NM_033183_1-18 HG1018484 SEQ. ID. NP_149439: leader sequence NO.:184 NM_033183_1-20 HG1018485 SEQ. ID. NP_149439: leader sequence NO.:185 NM_033183_1-16 HG1018487 SEQ. ID. NP_644808: leader sequence NO.:186 NM_139279_1-18 HG1018488 SEQ. ID. NP_644808: leader sequence NO.:187 NM_139279_1-20 HG1018489 SEQ. ID. NP_644808: leader sequence NO.:188 NM_139279_1-26 HG1018490 SEQ. ID. NP_644808: leader sequence NO.:189 NM_139279_1-23 HG1018492 SEQ. ID. NP_660295: leader sequence NO.:190 NM_145252_1-13 HG1018493 SEQ. ID. NP_660295: leader sequence NO.:191 NM_145252_1-16 HG1018494 SEQ. ID. NP_660295: leader sequence NO.:192 NM_145252_1-14 HG1018495 SEQ. ID. NP_660295: leader sequence NO.:193 NM_145252_1-17 HG1018497 SEQ. ID. NP_689534: HMM_SP leader sequenceNO.: 194 NM_152321_1-25 HG1018498 SEQ. ID. NP_689534: leader sequenceNO.: 195 NM_152321_1-21 HG1018500 SEQ. ID. NP_689848: HMM_SP leadersequence NO.: 196 NM_152635_1-18 HG1018501 SEQ. ID. NP_689848: leadersequence NO.: 197 NM_152635_1-16 HG1018502 SEQ. ID. NP_689848: leadersequence NO.: 198 NM_152635_1-15 HG1018504 SEQ. ID. NP_689968: HMM_SPleader sequence NO.: 199 NM_152755_1-21 HG1018506 SEQ. ID. NP_766630:HMM_SP leader sequence NO.: 200 NM_173042_1-29 HG1018507 SEQ. ID.NP_766630: leader sequence NO.: 201 NM_173042_1-24 HG1018508 SEQ. ID.NP_766630: leader sequence NO.: 202 NM_173042_1-28 HG1018510 SEQ. ID.NP_776214: HMM_SP leader sequence NO.: 203 NM_173842_1-23 HG1018511 SEQ.ID. NP_776214: leader sequence NO.: 204 NM_173842_1-25 HG1018513 SEQ.ID. NP_783165: HMM_SP leader sequence NO.: 205 NM_175575_1-32 HG1018514SEQ. ID. NP_783165: leader sequence NO.: 206 NM_175575_1-34 HG1018515SEQ. ID. NP_783165: leader sequence NO.: 207 NM_175575_1-29 HG1018516SEQ. ID. NP_783165: leader sequence NO.: 208 NM_175575_1-30 HG1018517SEQ. ID. NP_783165: leader sequence NO.: 209 NM_175575_1-27 HG1018857SEQ. ID. 27482680: HMM_SP leader sequence NO.: 210 27482679_1-26HG1018858 SEQ. ID. 27482680: leader sequence NO.: 211 27482679_1-24HG1015544 SEQ. ID. SEQ. ID. CLN00542945 hypothetical protein MGD- NO.:212 NO.: 235 CSF [Homo sapiens] untagged in vector pTT5. HG1019453 SEQ.ID. SEQ. ID. CLN00839395 hypothetical protein MGD- NO.: 213 NO.: 236 CSF[Homo sapiens] untagged in vector pTT2 HG1019454 SEQ. ID. SEQ. ID.CLN00732663 hypothetical protein MGD- NO.: 214 NO.: 237 CSF [Homosapiens] C- terminus V5H8 tagged in vector pTT5 HG1019455 SEQ. ID. SEQ.ID. CLN00840351 hypothetical protein MGD- NO.: 215 NO.: 238 CSF [Homosapiens] C- terminus V5H8 tagged in vector pTT2 HG1019456 SEQ. ID. SEQ.ID. CLN00758593 hypothetical protein MGD- NO.: 216 NO.: 239 CSF [Homosapiens] C- terminus V5H8 tagged in pIB/V5His-DEST vector (Invitrogen).HG1019457 SEQ. ID. SEQ. ID. CLN00848149 hypothetical protein MGD- NO.:217 NO.: 240 CSF [Homo sapiens]. Collagen SP(1- 23aa)_MGD-CSF(21 to241aa). Untagged in vector pTT5-G. HG1019458 SEQ. ID. SEQ. ID.CLN00821867 hypothetical protein MGD- NO.: 218 NO.: 241 CSF [Homosapiens]. MGD-CSF (1- 241aa)_TEV_V5 _StreptagII_H8. C- tagged in vectorpTT5-I. HG1019459 SEQ. ID. SEQ. ID. CLN00816424 hypothetical proteinMGD- NO.: 219 NO.: 242 CSF [Homo sapiens] Collagen SP (1-23aa)_MGD-CSF(21 to 241aa)_TEV_V5_StreptagII_H8. C- tagged in vectorpTT5-G HG1019460 SEQ. ID. SEQ. ID. CLN00816425 hypothetical protein MGD-NO.: 220 NO.: 243 CSF [Homo sapiens] Collagen SP(1-23aa)_H8_StreptagII_V5_TEV_MGD- CSF (21 to 241aa). N- tagged in vectorpTT5-H HG1019461 SEQ. ID. SEQ. ID. CLN00848160 hypothetical protein MGD-NO.: 221 NO.: 244 CSF [Homo sapiens]. Collagen SP(1- 23aa)_MGD-CSF(26 to241aa). Untagged in vector pTT5. HG1019462 SEQ. ID. SEQ. ID. CLN00848173hypothetical protein MGD- NO.: 222 NO.: 245 CSF [Homo sapiens]. CollagenSP(1- 23aa)_MGD-CSF(31 to 241aa). Untagged in vector pTT5. HG1019463SEQ. ID. SEQ. ID. CLN00848185 hypothetical protein MGD- NO.: 223 NO.:246 CSF [Homo sapiens] Collagen SP(1- 23aa)_MGD-CSF(21 to 213aa).Untagged in vector pTT5. HG1019464 SEQ. ID. SEQ. ID. CLN00848197hypothetical protein MGD- NO.: 224 NO.: 247 CSF [Homo sapiens] CollagenSP(1- 23aa)_MGD-CSF(21 to 231aa). Untagged in vector pTT5. HG1019465SEQ. ID. SEQ. ID. CLN00848209 hypothetical protein MGD- NO.: 225 NO.:248 CSF [Homo sapiens] Collagen SP(1- 23aa)_MGD-CSF(21 to 236aa).Untagged in vector pTT5. HG1019466 SEQ. ID. SEQ. ID. CLN00848220hypothetical protein MGD- NO.: 226 NO.: 249 CSF [Homo sapiens] CollagenSP(1- 23aa)_MGD-CSF(26 to 231aa). Untagged in vector pTT5. HG1019467SEQ. ID. SEQ. ID. CLN00840257 Phantom Clone NO.: 227 NO.: 250 2010004A03mouse ortholog of human MGC34647 cloned in vector pTT5. HG1019468 SEQ.ID. SEQ. ID. CLN00840253 Phantom Clone NO.: 228 NO.: 251 2010004A03mouse ortholog of human MGC34647. 12842044(1 to219aa)_TEV_V5_StreptagII_H8. cloned in vector pTT5-I HG1019469 SEQ. ID.SEQ. ID. CLN00847948 mouse ortholog of human NO.: 229 NO.: 252 MGC34647cloned in vector pTT5 235aa HG1019470 SEQ. ID. SEQ. ID. CLN00842712mouse ortholog of human NO.: 230 NO.: 253 MGC34647. 18921437(1-235aa)_TEV_V5_StreptagII_H8. cloned in pTT5-I HG1019471 SEQ. ID. SEQ.ID. NP_689669_(—) NO.: 231 NO.: 254 maturepeptide HG1019472 SEQ. ID.SEQ. ID. SEQ. ID. WO02048337_seq49 Incyte patent WO NO.: 232 NO.: 255NO.: 257 02/048337 (seqs 49/103) HG1019473 SEQ. ID. SEQ. ID.WO02048337_seq49_(—) NO.: 233 NO.: 256 maturepeptide HG1019474 SEQ. ID.Kozak_sequence GCCGCCACC NO.: 234 HG1019600 SEQ. ID. SEQ. ID.CLN00872284_20-241 CollagenSP_C35S- NO.: 258 NO.: 265 MGC34647(20 to241aa)_STP HG1019601 SEQ. ID. SEQ. ID. CLN00872342_20-241CollagenSP_C179S- NO.: 259 NO.: 266 MGC34647(20 to 241aa)_STP HG1019602SEQ. ID. SEQ. ID. CLN00873848_20-241 CollagenSP_C176S- NO.: 260 NO.: 267MGC34647(20 to 241aa)_STP HG1019603 SEQ. ID. SEQ. ID. CLN00873864_20-241CollagenSP_C190S- NO.: 261 NO.: 268 MGC34647(20 to 241aa)_STP HG1019604SEQ. ID. SEQ. ID. CLN00873948_20-241 CollagenSP_C167S- NO.: 262 NO.: 269MGC34647(20 to 241aa)_STP HG1019605 SEQ. ID. SEQ. ID. CLN00873956_20-241CollagenSP_C178S- NO.: 263 NO.: 270 MGC34647(20 to 241aa)_STP HG1019606SEQ. ID. SEQ. ID. CLN00873970_20-241 CollagenSP_C198S- NO.: 264 NO.: 271MGC34647(20 to 241aa)_STP

TABLE 2 Annotation of MGD-CSF FP ID HG1015544 HG1015545 HG1015546 CloneID MGD-CSF MGD- NP_689669 CSF_exon4 Pred Prot Len 241 53 242 Top HumanHit gi|22748957|ref| gi|22748957|ref| gi|22748957|ref| Accession NoNP_689669.1| NP_689669.1| NP_689669.1| Top Human Hit hypotheticalhypothetical hypothetical Annotation protein protein protein MGC34647MGC34647 MGC34647 [Homo sapiens] [Homo sapiens] [Homo sapiens] Top HumanHit Len 242 242  242 Match Len 241 53 242 Top Human Hit % ID 100 100 100 Over Query Len % ID Over Human Hit 100 22 100 Len

TABLE 3 Protein Coordinates of MGD-CSF FP ID HG1015544 HG1015545HG1015546 Clone ID MGD-CSF MGD-CSF_exon4 NP_689669 Cluster 190647 190647190647 Classification Secreted Secreted Secreted Pred Prot Len 241 53242 Treevote 0.93 0.75 0.92 Signal Peptide Coords (1-20)  (9-23) (1-20)Mature Protein Coords (21-241) (24-53) (21-241)

TABLE 4 Secretory Leader Sequence Annotations FP ID Source ID AnnotationHG1018265 collagen_leader_seq collagen alpha 1(IX) chain precursor, longsplice form - human HG1018268 112907: 21594845_1-17 Alpha-2-antiplasminprecursor (Alpha-2-plasmin inhibitor) HG1018269 112907: 21594845_1-13Alpha-2-antiplasmin precursor (Alpha-2-plasmin inhibitor) HG1018270112907: 21594845_1-19 Alpha-2-antiplasmin precursor (Alpha-2-plasmininhibitor) HG1018271 112907: 21594845_1-16 Alpha-2-antiplasmin precursor(Alpha-2-plasmin inhibitor) HG1018272 112907: 21594845_1-15Alpha-2-antiplasmin precursor (Alpha-2-plasmin inhibitor) HG101827413325208: 13325207_1-30 Trinucleotide repeat containing 5 [Homo sapiens]HG1018275 13325208: 13325207_1-25 Trinucleotide repeat containing 5[Homo sapiens] HG1018276 13325208: 13325207_1-33 Trinucleotide repeatcontaining 5 [Homo sapiens] HG1018277 13325208: 13325207_1-24Trinucleotide repeat containing 5 [Homo sapiens] HG1018278 13325208:13325207_1-26 Trinucleotide repeat containing 5 [Homo sapiens] HG101827913325208: 13325207_1-32 Trinucleotide repeat containing 5 [Homo sapiens]HG1018280 13325208: 13325207_1-27 Trinucleotide repeat containing 5[Homo sapiens] HG1018281 13325208: 13325207_1-23 Trinucleotide repeatcontaining 5 [Homo sapiens] HG1018282 13325208: 13325207_1-35Trinucleotide repeat containing 5 [Homo sapiens] HG1018284 13938307:13938306_1-24 ARMET protein [Homo sapiens] HG1018285 13938307:13938306_1-21 ARMET protein [Homo sapiens] HG1018287 14718453:14718452_1-19 calumenin [Homo sapiens] HG1018288 14718453: 14718452_1-15calumenin [Homo sapiens] HG1018289 14718453: 14718452_1-17 calumenin[Homo sapiens] HG1018291 15929966: 15929965_1-23 COL9A1 protein [Homosapiens] HG1018293 16356651: 16356650_1-21 NBL1 [Homo sapiens] HG101829416356651: 16356650_1-17 NBL1 [Homo sapiens] HG1018296 18204192:18204191_1-19 PACAP protein [Homo sapiens] HG1018297 18204192:18204191_1-22 PACAP protein [Homo sapiens] HG1018298 18204192:18204191_1-18 PACAP protein [Homo sapiens] HG1018299 18204192:18204191_1-16 PACAP protein [Homo sapiens] HG1018300 18204192:18204191_1-14 PACAP protein [Homo sapiens] HG1018302 23503038:15778555_1-20 Alpha-1B-glycoprotein precursor (Alpha-1-B glycoprotein)HG1018303 23503038: 15778555_1-16 Alpha-1B-glycoprotein precursor(Alpha-1-B glycoprotein) HG1018304 23503038: 15778555_1-21Alpha-1B-glycoprotein precursor (Alpha-1-B glycoprotein) HG101830627479535: 27479534_1-24 similar to Brain-specific angiogenesis inhibitor2 precursor [Homo sapiens] HG1018307 27479535: 27479534_1-20 similar toBrain-specific angiogenesis inhibitor 2 precursor [Homo sapiens]HG1018308 27479535: 27479534_1-26 similar to Brain-specific angiogenesisinhibitor 2 precursor [Homo sapiens] HG1018309 27479535: 27479534_1-21similar to Brain-specific angiogenesis inhibitor 2 precursor [Homosapiens] HG1018310 27479535: 27479534_1-23 similar to Brain-specificangiogenesis inhibitor 2 precursor [Homo sapiens] HG1018312 37182960:37182959_1-24 SPOCK2 [Homo sapiens] HG1018313 37182960: 37182959_1-19SPOCK2 [Homo sapiens] HG1018314 37182960: 37182959_1-22 SPOCK2 [Homosapiens] HG1018315 37182960: 37182959_1-20 SPOCK2 [Homo sapiens]HG1018316 37182960: 37182959_1-26 SPOCK2 [Homo sapiens] HG101831737182960: 37182959_1-21 SPOCK2 [Homo sapiens] HG1018319 7437388:1208426_1-24 protein disulfide-isomerase (EC 5341) ER60 precursor -human HG1018320 7437388: 1208426_1-23 protein disulfide-isomerase (EC5341) ER60 precursor - human HG1018322 NP_000286: NM_000295_1-24 serine(or cysteine) proteinase inhibitor, clade A (alpha-1 HG1018323NP_000286: NM_000295_1-18 serine (or cysteine) proteinase inhibitor,clade A (alpha-1 HG1018324 NP_000286: NM_000295_1-23 serine (orcysteine) proteinase inhibitor, clade A (alpha-1 HG1018325 NP_000286:NM_000295_1-17 serine (or cysteine) proteinase inhibitor, clade A(alpha-1 HG1018327 NP_000396: NM_000405_1-23 GM2 ganglioside activatorprecursor [Homo sapiens] HG1018328 NP_000396: NM_000405_1-18 GM2ganglioside activator precursor [Homo sapiens] HG1018329 NP_000396:NM_000405_1-25 GM2 ganglioside activator precursor [Homo sapiens]HG1018330 NP_000396: NM_000405_1-20 GM2 ganglioside activator precursor[Homo sapiens] HG1018331 NP_000396: NM_000405_1-21 GM2 gangliosideactivator precursor [Homo sapiens] HG1018333 NP_000495: NM_000504_1-23coagulation factor X precursor [Homo sapiens] HG1018334 NP_000495:NM_000504_1-19 coagulation factor X precursor [Homo sapiens] HG1018335NP_000495: NM_000504_1-20 coagulation factor X precursor [Homo sapiens]HG1018336 NP_000495: NM_000504_1-15 coagulation factor X precursor [Homosapiens] HG1018337 NP_000495: NM_000504_1-21 coagulation factor Xprecursor [Homo sapiens] HG1018338 NP_000495: NM_000504_1-17 coagulationfactor X precursor [Homo sapiens] HG1018340 NP_000573: NM_000582_1-18secreted phosphoprotein 1 (osteopontin, bone sialoprotein I, earlyHG1018341 NP_000573: NM_000582_1-16 secreted phosphoprotein 1(osteopontin, bone sialoprotein I, early HG1018342 NP_000573:NM_000582_1-15 secreted phosphoprotein 1 (osteopontin, bone sialoproteinI, early HG1018344 NP_000574: NM_000583_1-16 vitamin D-binding proteinprecursor [Homo sapiens] HG1018345 NP_000574: NM_000583_1-14 vitaminD-binding protein precursor [Homo sapiens] HG1018347 NP_000591:NM_000600_1-25 interleukin 6 (interferon, beta 2) [Homo sapiens]HG1018348 NP_000591: NM_000600_1-24 interleukin 6 (interferon, beta 2)[Homo sapiens] HG1018349 NP_000591: NM_000600_1-27 interleukin 6(interferon, beta 2) [Homo sapiens] HG1018351 NP_000598: NM_000607_1-18orosomucoid 1 precursor [Homo sapiens] HG1018353 NP_000604:NM_000613_1-19 hemopexin [Homo sapiens] HG1018354 NP_000604:NM_000613_1-25 hemopexin [Homo sapiens] HG1018355 NP_000604:NM_000613_1-21 hemopexin [Homo sapiens] HG1018356 NP_000604:NM_000613_1-23 hemopexin [Homo sapiens] HG1018357 NP_000604:NM_000613_1-31 hemopexin [Homo sapiens] HG1018359 NP_000726:NM_000735_1-26 glycoprotein hormones, alpha polypeptide precursor [Homosapiens] HG1018360 NP_000726: NM_000735_1-24 glycoprotein hormones,alpha polypeptide precursor [Homo sapiens] HG1018362 NP_000884:NM_000893_1-18 kininogen 1 [Homo sapiens] HG1018363 NP_000884:NM_000893_1-19 kininogen 1 [Homo sapiens] HG1018364 NP_000884:NM_000893_1-16 kininogen 1 [Homo sapiens] HG1018365 NP_000884:NM_000893_1-23 kininogen 1 [Homo sapiens] HG1018367 NP_000909:NM_000918_1-17 prolyl 4-hydroxylase, beta subunit [Homo sapiens]HG1018369 NP_000930: NM_000939_1-23 proopiomelanocortin [Homo sapiens]HG1018370 NP_000930: NM_000939_1-26 proopiomelanocortin [Homo sapiens]HG1018372 NP_000945: NM_000954_1-23 prostaglandin D2 synthase 21 kDa[Homo sapiens] HG1018373 NP_000945: NM_000954_1-22 prostaglandin D2synthase 21 kDa [Homo sapiens] HG1018374 NP_000945: NM_000954_1-18prostaglandin D2 synthase 21 kDa [Homo sapiens] HG1018376 NP_001176:NM_001185_1-18 alpha-2-glycoprotein 1, zinc [Homo sapiens] HG1018377NP_001176: NM_001185_1-20 alpha-2-glycoprotein 1, zinc [Homo sapiens]HG1018378 NP_001176: NM_001185_1-21 alpha-2-glycoprotein 1, zinc [Homosapiens] HG1018379 NP_001176: NM_001185_1-17 alpha-2-glycoprotein 1,zinc [Homo sapiens] HG1018381 NP_001266: NM_001275_1-18 chromogranin A[Homo sapiens] HG1018382 NP_001266: NM_001275_1-15 chromogranin A [Homosapiens] HG1018383 NP_001266: NM_001275_1-14 chromogranin A [Homosapiens] HG1018385 NP_001314: NM_001323_1-26 cystatin M precursor [Homosapiens] HG1018386 NP_001314: NM_001323_1-18 cystatin M precursor [Homosapiens] HG1018387 NP_001314: NM_001323_1-20 cystatin M precursor [Homosapiens] HG1018388 NP_001314: NM_001323_1-28 cystatin M precursor [Homosapiens] HG1018389 NP_001314: NM_001323_1-21 cystatin M precursor [Homosapiens] HG1018390 NP_001314: NM_001323_1-23 cystatin M precursor [Homosapiens] HG1018392 NP_001822: NM_001831_1-22 clusterin isoform 1 [Homosapiens] HG1018393 NP_001822: NM_001831_1-18 clusterin isoform 1 [Homosapiens] HG1018394 NP_001822: NM_001831_1-14 clusterin isoform 1 [Homosapiens] HG1018396 NP_002206: NM_002215_1-24 inter-alpha (globulin)inhibitor H1 [Homo sapiens] HG1018397 NP_002206: NM_002215_1-29inter-alpha (globulin) inhibitor H1 [Homo sapiens] HG1018398 NP_002206:NM_002215_1-30 inter-alpha (globulin) inhibitor H1 [Homo sapiens]HG1018399 NP_002206: NM_002215_1-23 inter-alpha (globulin) inhibitor H1[Homo sapiens] HG1018400 NP_002206: NM_002215_1-31 inter-alpha(globulin) inhibitor H1 [Homo sapiens] HG1018402 NP_002300:NM_002309_1-22 leukemia inhibitory factor (cholinergic differentiationfactor) HG1018403 NP_002300: NM_002309_1-23 leukemia inhibitory factor(cholinergic differentiation factor) HG1018405 NP_002336: NM_002345_1-18lumican [Homo sapiens] HG1018406 NP_002336: NM_002345_1-15 lumican [Homosapiens] HG1018407 NP_002336: NM_002345_1-17 lumican [Homo sapiens]HG1018408 NP_002336: NM_002345_1-14 lumican [Homo sapiens] HG1018410NP_002402: NM_002411_1-18 secretoglobin, family 2A, member 2 [Homosapiens] HG1018412 NP_002505: NM_002514_1-30 nov precursor [Homosapiens] HG1018413 NP_002505: NM_002514_1-32 nov precursor [Homosapiens] HG1018414 NP_002505: NM_002514_1-28 nov precursor [Homosapiens] HG1018415 NP_002505: NM_002514_1-27 nov precursor [Homosapiens] HG1018416 NP_002505: NM_002514_1-31 nov precursor [Homosapiens] HG1018418 NP_002892: NM_002901_1-26 reticulocalbin 1 precursor[Homo sapiens] HG1018419 NP_002892: NM_002901_1-22 reticulocalbin 1precursor [Homo sapiens] HG1018420 NP_002892: NM_002901_1-29reticulocalbin 1 precursor [Homo sapiens] HG1018421 NP_002892:NM_002901_1-24 reticulocalbin 1 precursor [Homo sapiens] HG1018422NP_002892: NM_002901_1-23 reticulocalbin 1 precursor [Homo sapiens]HG1018424 NP_002893: NM_002902_1-25 reticulocalbin 2, EF-hand calciumbinding domain [Homo sapiens] HG1018425 NP_002893: NM_002902_1-19reticulocalbin 2, EF-hand calcium binding domain [Homo sapiens]HG1018426 NP_002893: NM_002902_1-22 reticulocalbin 2, EF-hand calciumbinding domain [Homo sapiens] HG1018427 NP_002893: NM_002902_1-18reticulocalbin 2, EF-hand calcium binding domain [Homo sapiens]HG1018428 NP_002893: NM_002902_1-20 reticulocalbin 2, EF-hand calciumbinding domain [Homo sapiens] HG1018429 NP_002893: NM_002902_1-21reticulocalbin 2, EF-hand calcium binding domain [Homo sapiens]HG1018430 NP_002893: NM_002902_1-23 reticulocalbin 2, EF-hand calciumbinding domain [Homo sapiens] HG1018432 NP_005133: NM_005142_1-19gastric intrinsic factor (vitamin B synthesis) [Homo sapiens] HG1018433NP_005133: NM_005142_1-18 gastric intrinsic factor (vitamin B synthesis)[Homo sapiens] HG1018434 NP_005133: NM_005142_1-20 gastric intrinsicfactor (vitamin B synthesis) [Homo sapiens] HG1018435 NP_005133:NM_005142_1-24 gastric intrinsic factor (vitamin B synthesis) [Homosapiens] HG1018436 NP_005133: NM_005142_1-16 gastric intrinsic factor(vitamin B synthesis) [Homo sapiens] HG1018437 NP_005133: NM_005142_1-17gastric intrinsic factor (vitamin B synthesis) [Homo sapiens] HG1018438NP_005133: NM_005142_1-14 gastric intrinsic factor (vitamin B synthesis)[Homo sapiens] HG1018440 NP_005445: NM_005454_1-17 cerberus 1 [Homosapiens] HG1018442 NP_005555: NM_005564_1-18 lipocalin 2 (oncogene 24p3)[Homo sapiens] HG1018443 NP_005555: NM_005564_1-20 lipocalin 2 (oncogene24p3) [Homo sapiens] HG1018444 NP_005555: NM_005564_1-15 lipocalin 2(oncogene 24p3) [Homo sapiens] HG1018446 NP_005690: NM_005699_1-29interleukin 18 binding protein isoform C precursor [Homo sapiens]HG1018447 NP_005690: NM_005699_1-24 interleukin 18 binding proteinisoform C precursor [Homo sapiens] HG1018448 NP_005690: NM_005699_1-28interleukin 18 binding protein isoform C precursor [Homo sapiens]HG1018450 NP_006560: NM_006569_1-19 cell growth regulator with EF handdomain 1 [Homo sapiens] HG1018451 NP_006560: NM_006569_1-18 cell growthregulator with EF hand domain 1 [Homo sapiens] HG1018452 NP_006560:NM_006569_1-21 cell growth regulator with EF hand domain 1 [Homosapiens] HG1018454 NP_006856: NM_006865_1-15 leukocyteimmunoglobulin-like receptor, subfamily A (without TM HG1018456NP_036577: NM_012445_1-26 spondin 2, extracellular matrix protein [Homosapiens] HG1018457 NP_036577: NM_012445_1-25 spondin 2, extracellularmatrix protein [Homo sapiens] HG1018458 NP_036577: NM_012445_1-24spondin 2, extracellular matrix protein [Homo sapiens] HG1018459NP_036577: NM_012445_1-28 spondin 2, extracellular matrix protein [Homosapiens] HG1018461 NP_055070: NM_014255_1-20 transmembrane protein 4[Homo sapiens] HG1018462 NP_055070: NM_014255_1-18 transmembrane protein4 [Homo sapiens] HG1018463 NP_055070: NM_014255_1-16 transmembraneprotein 4 [Homo sapiens] HG1018465 NP_055582: NM_014767_1-24sparc/osteonectin, cwcv and kazal-like domains proteoglycan HG1018466NP_055582: NM_014767_1-19 sparc/osteonectin, cwcv and kazal-like domainsproteoglycan HG1018467 NP_055582: NM_014767_1-22 sparc/osteonectin, cwcvand kazal-like domains proteoglycan HG1018468 NP_055582: NM_014767_1-20sparc/osteonectin, cwcv and kazal-like domains proteoglycan HG1018469NP_055582: NM_014767_1-26 sparc/osteonectin, cwcv and kazal-like domainsproteoglycan HG1018470 NP_055582: NM_014767_1-21 sparc/osteonectin, cwcvand kazal-like domains proteoglycan HG1018472 NP_055697: NM_014882_1-18Rho GTPase activating protein 25 isoform b [Homo sapiens] HG1018474NP_056965: NM_015881_1-18 dickkopf homolog 3 [Homo sapiens] HG1018475NP_056965: NM_015881_1-19 dickkopf homolog 3 [Homo sapiens] HG1018476NP_056965: NM_015881_1-22 dickkopf homolog 3 [Homo sapiens] HG1018477NP_056965: NM_015881_1-16 dickkopf homolog 3 [Homo sapiens] HG1018478NP_056965: NM_015881_1-21 dickkopf homolog 3 [Homo sapiens] HG1018480NP_057603: NM_016519_1-26 ameloblastin precursor [Homo sapiens]HG1018481 NP_057603: NM_016519_1-28 ameloblastin precursor [Homosapiens] HG1018483 NP_149439: NM_033183_1-18 chorionic gonadotropin,beta polypeptide 8 recursor [Homo sapiens] HG1018484 NP_149439:NM_033183_1-20 chorionic gonadotropin, beta polypeptide 8 recursor [Homosapiens] HG1018485 NP_149439: NM_033183_1-16 chorionic gonadotropin,beta polypeptide 8 recursor [Homo sapiens] HG1018487 NP_644808:NM_139279_1-18 multiple coagulation factor deficiency 2 [Homo sapiens]HG1018488 NP_644808: NM_139279_1-20 multiple coagulation factordeficiency 2 [Homo sapiens] HG1018489 NP_644808: NM_139279_1-26 multiplecoagulation factor deficiency 2 [Homo sapiens] HG1018490 NP_644808:NM_139279_1-23 multiple coagulation factor deficiency 2 [Homo sapiens]HG1018492 NP_660295: NM_145252_1-13 similar to common salivary protein 1[Homo sapiens] HG1018493 NP_660295: NM_145252_1-16 similar to commonsalivary protein 1 [Homo sapiens] HG1018494 NP_660295: NM_145252_1-14similar to common salivary protein 1 [Homo sapiens] HG1018495 NP_660295:NM_145252_1-17 similar to common salivary protein 1 [Homo sapiens]HG1018497 NP_689534: NM_152321_1-25 hypothetical protein FLJ32115 [Homosapiens] HG1018498 NP_689534: NM_152321_1-21 hypothetical proteinFLJ32115 [Homo sapiens] HG1018500 NP_689848: NM_152635_1-18oncoprotein-induced transcript 3 [Homo sapiens] HG1018501 NP_689848:NM_152635_1-16 oncoprotein-induced transcript 3 [Homo sapiens] HG1018502NP_689848: NM_152635_1-15 oncoprotein-induced transcript 3 [Homosapiens] HG1018504 NP_689968: NM_152755_1-21 hypothetical proteinMGC40499 [Homo sapiens] HG1018506 NP_766630: NM_173042_1-29 interleukin18 binding protein isoform A precursor [Homo sapiens] HG1018507NP_766630: NM_173042_1-24 interleukin 18 binding protein isoform Aprecursor [Homo sapiens] HG1018508 NP_766630: NM_173042_1-28 interleukin18 binding protein isoform A precursor [Homo sapiens] HG1018510NP_776214: NM_173842_1-23 interleukin 1 receptor antagonist isoform 1precursor [Homo sapiens] HG1018511 NP_776214: NM_173842_1-25 interleukin1 receptor antagonist isoform 1 precursor [Homo sapiens] HG1018513NP_783165: NM_175575_1-32 WFIKKN2 protein [Homo sapiens] HG1018514NP_783165: NM_175575_1-34 WFIKKN2 protein [Homo sapiens] HG1018515NP_783165: NM_175575_1-29 WFIKKN2 protein [Homo sapiens] HG1018516NP_783165: NM_175575_1-30 WFIKKN2 protein [Homo sapiens] HG1018517NP_783165: NM_175575_1-27 WFIKKN2 protein [Homo sapiens] HG101885727482680: 27482679_1-26 similar to hypothetical protein 9330140G23 [Homosapiens] HG1018858 27482680: 27482679_1-24 similar to hypotheticalprotein 9330140G23 [Homo sapiens]

TABLE 5 MGD-CSF Construct Annotations Vector Clone ID AnnotationDescription Tag CLN00542945 hypothetical protein MGD-CSF pTT5-Gateway notag [Homo sapiens] CLN00732663 hypothetical protein MGD-CSF pTT5-GatewayC-Tagged (V5H8) [Homo sapiens] CLN00839395 hypothetical protein MGD-CSFpTT2-Gateway C-Tagged (V5H8) [Homo sapiens] CLN00840351 hypotheticalprotein MGD-CSF pTT2-Gateway C-Tagged (V5H8) [Homo sapiens] CLN00843208hypothetical protein MGD-CSF ptt2-I no tag [Homo sapiens] CLN00758593hypothetical protein MGD-CSF pIB C-Tagged (V5H8) [Homo sapiens]CLN00848149 SP_MGD-CSF(21 to 241aa) pTT5-G no tag Cleavable C-Tagged andN- Tagged Constructs CLN00816424 SP_MGD-CSF(21 to pTT5-G C-Tagged241aa)_TEV_V5_StrecTagII_H8 (TEV_V5_StrecTagII_H8) CLN00816425SP_TEV_V5_StrecTagII_H8_MGD- pTT5-H N-Tagged CSF(21 to 241aa)(SP_TEV_V5_StrecTagII_H8) CLN00821867 MGD-CSF(1- pTT5-I C-Tagged241)_TEV_V5_StrecTagII_H8 (TEV_V5_StrecTagII_H8) Mouse OrthologuesCLN00840253 hypothetical protein MGC34647 pTT5-I C-Tagged [mouse 660 bp](TEV_V5_StrecTagII_H8) CLN00840257 hypothetical protein MGC34647 pTT5-Ino tag [mouse 660 bp] CLN00842712 hypothetical protein MGC34647 pTT5-IC-Tagged [mouse 708 bp] (TEV_V5_StrecTagII_H8) CLN00847948 hypotheticalprotein MGC34647 pTT5-I no tag [mouse 708 bp] Deletion mutantsCLN00848160 SP_MGD-CSF(26 to 241aa) pTT5-G no tag CLN00848173 MGD-CSF(31to 241aa) pTT5-G no tag CLN00848185 SP_MGD-CSF(21 to 213aa) pTT5-G notag CLN00848197 SP_MGD-CSF(21 to 231aa) pTT5-G no tag CLN00848209SP_MGD-CSF(21 to 236aa) pTT5-G no tag CLN00848220 SP_MGD-CSF(26 to231aa) pTT5-G no tag

TABLE 6 MGD-CSF Promotes Myelocytic Cell Proliferation In Vitro Clone IDDescription Potency Expression CLN00542945 MGD-CSF (1-241 aa) +++ +++CLN00848149 CSP-025 (20 to 241aa) ++ ++ CLN00848160 CSP-025 (25 to241aa) + + CLN00848173 CSP-025 (30 to 241aa) ++ ++ CLN00848185 CSP-025(20 to 213 aa) +++ ++ CLN00848197 CSP-025 (20 to 231aa) ++ +++CLN00848209 CSP-025 (20 to 236 aa) + + CLN00848220 CSP-025 (25 to 231aa)++ ++ Vector Control −

TABLE 7 MGD-CSF Promotes Myelocytic Proliferation In Vivo Animal IDDescription Monocytes/ul A: Human MGD-CSF Promotes Myelocytic CellProliferation in Mice In Vivo 1 Vector control 94.0 2 Vector control84.0 3 Vector control 52.0 4 human MGD-CSF 216.0 5 human MGD-CSF 0.0 6human MGD-CSF 268.0 B: Mouse MGD-CSF Promotes Myelocytic CellProliferation in Mice In Vivo 1 Vector control 0.0 2 Vector control 0.03 Vector control 0.0 4 Vector control 0.0 5 Vector control 0.0 6 Vectorcontrol 0.0 7 mouse MGD-CSF 50.0 8 mouse MGD-CSF 0.0 9 mouse MGD-CSF61.0 10 mouse MGD-CSF 78.0 11 mouse MGD-CSF 0.0 12 mouse MGD-CSF 77.0

TABLE 8 MGD-CSF Gene Expression MGC34647 Total Gene Disease PathologyPositive Logic % Total Myelodysplastic Refractory anemia 4 4 100% syndrome with excess blasts Myelodysplastic Refractory anemia 1 1 100% syndrome with ringed sideroblasts Myelodysplastic Refractory anemia 2 367% syndrome with excess blasts in transformation MyelodysplasticMyelodysplastic 1 2 50% syndrome syndrome (morphologic abnormality)Acute B-cell Precursor B-cell 3 6 50% lymphoblastic lymphoblasticleukemia leukemia Acute myeloid Acute myeloid 1 4 25% leukemia leukemia,with maturation Acute myeloid Acute myeloid 2 11 18% leukemia leukemia,without maturation Acute myeloid Acute myeloid 1 7 14% leukemialeukemia, minimal differentiation Chronic myeloid Chronic myeloid 2 45 4% leukemia leukemia Chronic Chronic 1 40  3% lymphocytic lymphocyticleukemia leukemia Acute Acute 1 36  3% promyelocytic promyelocytic (3from (33% from leukemia leukemia bone marrow) bone marrow)

1-139. (canceled)
 140. A method of increasing the number of humanmonocytes comprising contacting human monocytes with a recombinantpolypeptide comprising an amino acid sequence that is at least 95%identical to the amino acid sequence of SEQ ID NO:9, wherein thepolypeptide increases the number of monocytes in a mouse model ofmonocyte proliferation.
 141. The method of claim 140, wherein thepolypeptide further comprises a fusion partner.
 142. The method of claim141, wherein the fusion partner is a succinyl group.
 143. The method ofclaim 141, wherein the fusion partner is selected from a polymer and asecond polypeptide.
 144. The method of claim 143, wherein the polymer ispolyethylene glycol.
 145. The method of claim 143, wherein the secondpolypeptide is selected from a serum albumin, a fetuin A, a fetuin B, aleucine zipper domain, a tetranectin trimerization domain, a mannosebinding protein, and an Fc.
 146. The method of claim 145, wherein thefusion partner is an Fc.
 147. The method of claim 140, wherein thepolypeptide comprises the amino acid sequence of SEQ ID NO:
 9. 148. Themethod of claim 147, wherein the polypeptide further comprises a fusionpartner.
 149. The method of claim 148, wherein the fusion partner is asuccinyl group.
 150. The method of claim 148, wherein the fusion partneris selected from a polymer and a second polypeptide.
 151. The method ofclaim 150, wherein the polymer is polyethylene glycol.
 152. The methodof claim 150, wherein the second polypeptide is selected from a serumalbumin, a fetuin A, a fetuin B, a leucine zipper domain, a tetranectintrimerization domain, a mannose binding protein, and an Fc.
 153. Themethod of claim 152, wherein the fusion partner is an Fc.
 154. Themethod of claim 150, wherein the polypeptide is produced in host cellsthat comprise a polynucleotide encoding the polypeptide.
 155. The methodof claim 154, wherein the host cells are a 293 cell line.
 156. Themethod of claim 154, wherein the host cells are a 293-6E cell line. 157.The method of claim 154, wherein the host cells are a Chinese hamsterovary (CHO) cell line.
 158. The method of claim 154, wherein thepolynucleotide comprises the nucleic acid sequence of SEQ ID NO: 3.